Dissecting Adipose Tissue Lipolysis: Molecular Regulation and Implications for Metabolic Disease

T S NIELSEN and others Dissecting adipose tissue 52:3 R199–R222 Review lipolysis

Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease

Thomas Svava Nielsen1,2, Niels Jessen2,3, Jens Otto L Jørgensen2, Niels Møller2 and Sten Lund2

1The Novo Nordisk Foundation Center for Basic Metabolic Research, Section on Integrative Physiology, Faculty of Correspondence Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 6.6.30, DK-2200 N Copenhagen, Denmark should be addressed 2Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Nørrebrogade 44, Bldg. 3.0, to T S Nielsen 8000 Aarhus C, Denmark Email 3Department of Molecular Medicine, Aarhus University Hospital, Brendstrupga˚ rdsvej 100, 8200 Aarhus N, Denmark [email protected]

Abstract

Lipolysis is the process by which triglycerides (TGs) are hydrolyzed to free fatty acids (FFAs) Key Words and glycerol. In adipocytes, this is achieved by sequential action of adipose TG lipase (ATGL), " lipolysis hormone-sensitive lipase (HSL), and monoglyceride lipase. The activity in the lipolytic " adipose tissue pathway is tightly regulated by hormonal and nutritional factors. Under conditions of " ATGL negative energy balance such as fasting and exercise, stimulation of lipolysis results in a " HSL profound increase in FFA release from adipose tissue (AT). This response is crucial in order to " free fatty acids provide the organism with a sufﬁcient supply of substrate for oxidative metabolism. " type 2 diabetes However, failure to efﬁciently suppress lipolysis when FFA demands are low can have serious metabolic consequences and is believed to be a key mechanism in the development of type 2

Journal of Molecular Endocrinology diabetes in obesity. As the discovery of ATGL in 2004, substantial progress has been made in the delineation of the remarkable complexity of the regulatory network controlling adipocyte lipolysis. Notably, regulatory mechanisms have been identified on multiple levels of the lipolytic pathway, including gene transcription and translation, post-translational modifications, intracellular localization, protein–protein interactions, and protein stability/ degradation. Here, we provide an overview of the recent advances in the field of AT lipolysis with particular focus on the molecular regulation of the two main lipases, ATGL and HSL, and Journal of Molecular the intracellular and extracellular signals affecting their activity. Endocrinology (2014) 52, R199–R222

Introduction

The major energy reserve in mammals consists of fat stored (FFAs). These are delivered to peripheral tissues where they in adipose tissue (AT). In periods of excess energy intake, can serve as substrate for b-oxidation and ATP production. dietary lipids are taken up by fat cells (adipocytes) in AT Only adipocytes have the ability to secrete FFAs into the and esteriﬁed into triglycerides (TGs), which are stored in circulation (Kolditz & Langin 2010). Hence, in the post- cytosolic lipid droplets (LDs). In conditions like fasting absorptive state and during physical exercise, the vast and exercise, when mobilization of endogenous energy majority of the systemic FFA originates from AT (Jensen stores is required, TG is hydrolyzed through the process of 2003). The unique ability of AT to balance storage and lipolysis and released to the circulation as free fatty acids release of lipids in response to altered nutrient demands

http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JME-13-0277 Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 03:02:49PM via free access Review T S NIELSEN and others Dissecting adipose tissue 52:3 R200 lipolysis

provides the organism with an FFA-buffering system of and oral glucose tolerance can be obtained by pharma- essentially unlimited capacity (Frayn 2002). However, the cological reductions of chronically elevated plasma FFA metabolic consequences of an excessive expansion of AT levels both in type 2 diabetic patients, obese nondiabetic are considerable. In humans, obesity is closely associated subjects, and nondiabetic subjects genetically predisposed with numerous risk factors that constitute the so-called to type 2 diabetes (Santomauro et al. 1999, Cusi et al. metabolic syndrome. This includes abdominal obesity, 2007). Accordingly, excessive FFA mobilization from AT hypertension, dyslipidemia,andglucoseintolerance, is widely conceived as playing a pivotal role in insulin which are key elements in the pathogenesis of cardiovas- resistance and type 2 diabetes, suggesting that dysregula- cular disease and type 2 diabetes (Alberti et al. 2009). The tion of AT lipolysis in the obese state is a contributing physiological link between obesity and metabolic disease factor to the development of metabolic disease. in humans is currently not completely understood, and one of the great enigmas is the remarkable individual differences in the predisposition to obesity-induced Major signaling pathways in AT lipolysis metabolic disease. This has led to the proposal of the – Lipolysis is the sequential hydrolysis of one TG molecule AT expandability hypothesis – (Virtue & Vidal-Puig 2010, into three FFAs and one glycerol by a class of hydrolytic Hardy et al. 2012), which states that the capacity of AT to enzymes commonly known as lipases. In mammalian expand appropriately when lipid storage is needed is lipolysis, three lipases act in sequence with the concomi- limited for a given individual. Hence, when the limit is tant release of one FFA in each step (Fig. 1); adipose TG exceeded, lipids begin to accumulate in ectopic tissues lipase (ATGL) converts TG to DG and is the rate-limiting causing metabolic dysfunction and insulin resistance due enzyme in the lipolytic pathway (Zimmermann et al. to lipotoxic effects. An emerging view is that this 2004). DG is hydrolyzed to MG by hormone-sensitive lipotoxicity is likely not caused by excess TG in itself, lipase (HSL; Haemmerle et al. 2002), and monoglyceride but rather by an excess of lipid intermediates and lipase (MGL) cleaves MG into glycerol and FFA (Fredrikson metabolites released from hypertrophic adipocytes. et al. 1986). The major positive regulators of human Several recent reviews have addressed these mechanisms lipolysis are catecholamines and natriuretic peptides in detail (Boura-Halfon & Zick 2009, Virtue & Vidal-Puig (NPs), while antilipolysis primarily is mediated by insulin 2010, Copps & White 2012, Hardy et al. 2012, Zechner and catecholamines. et al. 2012, Czech et al. 2013). Notably, it is now clear that besides serving as energy-dense metabolic substrates,

Journal of Molecular Endocrinology most, if not all, lipolytic products and intermediates like Catecholamines diacylglycerol (DG), monoacylglycerol (MG), and FFA The catecholamines, and specifically the stress hormones (and metabolites derived from these) play essential roles adrenaline and noradrenaline, are the primary mediators in multiple signaling pathways, both at the systemic and of adrenergic signaling in AT. The manner by which at the intracellular level (Zechner et al. 2012). When catecholamines regulate lipolysis is unusual as these present in excess, several of these lipid intermediates have hormones are able to both stimulate and inhibit lipolysis been suggested to induce insulin resistance in ectopic depending on their relative affinity for different adrenergic tissues by interfering with insulin signaling at the level of receptors (ARs). Thus, stimulation of lipolysis requires the the insulin receptor substrate (IRS) proteins (Boura-Halfon activation of b-ARs on the surface of the adipocyte, while & Zick 2009, Copps & White 2012). antilipolytic signals are transmitted by the a2-AR Being the major lipid species released from AT, FFA is likely one of the key elements in ectopic lipid accumu- ATGL HSL MGL lation and lipotoxicity. Thus, experimental elevation of TG DG MG Glycerol plasma FFA levels in human subjects acutely and dose FFA FFA FFA dependently counteracts peripheral insulin-stimulated glucose uptake and oxidation (Belfort et al. 2005, Gormsen Figure 1 et al. 2007, Hoeg et al. 2011). Furthermore, high FFA levels Schematic illustration of the lipolytic pathway. To fully hydrolyze TGs, attenuate the insulin-mediated suppression of hepatic ATGL, HSL, and MGL act in sequence, with the release of one FFA in each glucose production contributing to the impairment of step. This successively converts TG to DG, then to MG, and finally to glycerol and a total of tree FFA. TG, triglyceride; ATGL, adipose TG lipase; DG, whole-body glucose tolerance (Roden et al. 2000). Consist- diacylglycerol; FFA, free fatty acid; HSL, hormone-sensitive lipase; MG, ently, improvements in whole-body insulin sensitivity monoacylglycerol; MGL, monoglyceride lipase.

(Robidoux et al. 2004; Fig. 2). Three different b-AR Garton et al. 1988, Anthonsen et al. 1998). Phosphoryl-

subtypes exist (b1, b2, and b3), but in humans, only the ation of PLIN1 promotes the release of comparative gene

b1 and b2 isoforms are involved in lipolysis (Mauriege et al. identiﬁcation-58 (CGI-58), which is a potent co-activator

1988, Barbe et al. 1996, Tavernier et al. 1996). Both a2-AR of ATGL (Lass et al. 2006, Granneman et al. 2009). This and b-AR belong to the G-protein-coupled receptor facilitates the activation of ATGL, thus initiating the

(GPCR) family: the G-protein associated with a2-AR stimulated lipolytic cascade. Furthermore, PKA-mediated

contain the inhibitory Gi subunit, while b-AR-associated phosphorylation of HSL causes a rapid activation and

G-proteins contain the stimulating Gs subunit (Lafontan translocation of the lipase from the cytosol to the surface & Berlan 1993). The activation of the receptors causes the of the LDs (Egan et al. 1992). Here, it docks on the G-proteins to interact with adenylyl cyclase (AC), which phosphorylated PLIN1 and thereby gains access to its DG

is inhibited by interaction with Gi and activated by substrate, which is being generated by ATGL (Shen et al.

interaction with Gs (Lafontan & Berlan 1993). Upon 2009, Wang et al. 2009). activation, AC converts ATP to cAMP, resulting in an increase in intracellular cAMP levels, which activates Natriuretic peptides protein kinase A (PKA, also known as cAMP-dependent protein kinase; Langin 2006). Activated PKA phosphory- In addition to catecholamines, the cardiac hormones lates the LD-associated protein PLIN1 (Greenberg atrial NP (ANP) and B-type NP (BNP) are important et al. 1991) and cytoplasmic HSL (Stralfors et al. 1984, positive regulators of AT lipolysis in humans. NPs, which

α β 2-AR 1/2-AR NPR-A

AC GC Gi PDK Gs Cytosol ATP GTP cAMP cGMP PIP3 PKB/Akt PDE3B PDE5 PI3K PKA PKG

Journal of Molecular Endocrinology 5′-AMP ′ PIP2 5 -GMP IR IRS1/2 HSL Three FFA + glycerol CGI-58 P P P P P P P HSL P P PLIN1 MGL P PLIN1 P ATGL PLIN1 P DG MG

CGI-58 TG Lipid droplet

Figure 2 Primary signaling pathways in human lipolysis. Black and red lines indicate PDE5-mediated conversion of cGMP to 50-GMP, although the upstream pro-lipolytic and anti-lipolytic signaling events, respectively. Arrows signals regulating this process are currently unknown. The dashed line indicate stimulation and/or translocation and blunt lines indicate inhi- indicates a putative Akt-independent insulin pathway acting selectively

bition. Stimulation of lipolysis is dependent on PKA- or PKG-mediated on PLIN1. a2-ARs, a2-adrenergic receptors; AC, adenylyl cyclase; TG,

phosphorylation of HSL and PLIN1. PKG is activated by cGMP, which is triglyceride; ATGL, adipose TG lipase; b1/2-ARs, b1- and b2-adrenergic increased in response to activation of the GC-coupled NPR-A. Similarly, receptors; CGI-58, comparative gene identiﬁcation-58; DG, diacylglycerol;

stimulation of the Gs-protein-coupled b1/2-ARs activates AC, which FFA, free fatty acid; GC, guanylyl cyclase; HSL, hormone-sensitive lipase; IR,

generates cAMP and activates PKA. Conversely, activation of Gi-protein- insulin receptor; IRS1/2, IR substrates 1 and 2; MG, monoacylglycerol; MGL,

coupled a2-ARs inhibits AC and thereby reduces cAMP-dependent signaling monoglyceride lipase; NPR-A, type-A natriuretic peptide receptor; PDE3B, to lipolysis. Stimulation of the insulin signaling pathway through the IR phosphodiesterase 3B; PDK, phosphoinositide-dependent kinase; PI3K, increases the activity of PDE3B, which converts cAMP to 50-AMP, thus phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB/Akt, protein decreasing PKA activity and suppressing lipolysis. PKG activity is reduced by kinase B; PLIN1, perilipin 1.

are released from the atrial and ventricular walls of the rat adipocytes by a cAMP-independent mechanism heart in response to myotube distension (Clerico et al. (Londos et al. 1985). By carefully measuring PKA activity 2011), stimulate the guanylyl cyclase (GC)-linked type-A ratios in response to increasing concentrations of iso- NP receptor on the adipocytes (Sengenes et al. 2000) prenaline (a b-AR agonist), the authors found that when (Fig. 2). Accordingly, upon stimulation of the receptor, GC insulin was added to the cells at submaximal lipolytic converts intracellular GTP into cGMP resulting in the stimulation, the insulin-mediated reduction in PKA activation of PKG (also known as the cGMP-dependent activity was not sufficient to explain the resulting drop protein kinase), and, just like PKA, this kinase activates the in lipolysis. Conversely, under conditions of maximal lipolytic cascade by phosphorylation of PLIN1 and HSL lipolysis, insulin-mediated changes in PKA activity could (Sengenes et al. 2003). However, despite the similarities fully account for the resulting change in lipolytic rates. between PKA- and PKG-mediated lipolysis, they are A recent study in 3T3-L1 adipocytes has partially distinct pathways and, unlike the cAMP-dependent delineated this bimodal insulin effect by showing that pathway, NP-mediated lipolysis is unresponsive to the insulin-mediated antilipolysis at submaximal b-AR stimu- antilipolytic effects of phosphodiesterase 3B (PDE3B; lation (but not at maximal stimulation) can proceed Sengenes et al.2000, Moro et al. 2004a). Instead, through an alternative PI3K-dependent pathway that is counter-regulation of the NP pathway is believed to independent of Akt (Choi et al. 2010). Acting through as occur by hydrolysis of cGMP by other members of the yet unidentified downstream effectors, the pathway was PDE family of PDEs (Armani et al. 2011). Indeed, PDE5 shown to inhibit lipolysis by selectively reducing PLIN1 expression and activity has been found in isolated human phosphorylation without affecting the phosphorylation adipocytes from both subcutaneous AT (Moro et al. 2007a) status of HSL (Fig. 2). Considering the key role of PLIN1 in and visceral AT (Aversa et al. 2011); however, this enzyme the regulation of ATGL- and HSL-mediated lipolysis such a appears to be insufficient to control ANP-mediated pathway would indeed be expected to have a substantial lipolysis (Moro et al. 2007a). Hence, at present, the details impact on overall lipolytic rates. of the regulatory pathways counteracting the lipolytic Besides the inhibitory effects on the lipolytic pathway, action of NPs in vivo remains poorly understood (Armani insulin also promotes lipid storage by activating a range of et al. 2011). pathways involved in the uptake, synthesis, and storage of TG in adipocytes. A comprehensive review of these lipogenic effects of insulin has been published recently Insulin (Czech et al. 2013).

Journal of Molecular Endocrinology Lipolysis is exceptionally sensitive to the action of insulin (Jensen & Nielsen 2007), which constitutes the major Alternative regulatory pathways antilipolytic pathway in human lipolysis (Fig. 2). The IR possesses intrinsic tyrosine kinase activity. Thus, binding Although catecholamines, insulin, and NPs represent the of insulin induces IR autophosphorylation and sub- major regulators of human lipolysis, several other factors sequent phosphorylation of the IRS1/2 (White 1998). can modulate lipolysis in AT, either directly by receptor- This promotes the activation of phosphatidylinositol mediated signaling or indirectly by remodeling of the 3-kinase (PI3K), which converts phosphatidylinositol- lipolytic cascade. Figure 3 shows an overview of the

4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5- different alternative pathways described below.

triphosphate (PIP3)(Whitman et al. 1988, Carpenter et al. 1990). Generation of PIP activates the phosphoinositide- 3 Agents acting through cAMP-dependent signaling dependent kinase causing phosphorylation and activation of Akt (also known as PKB; Alessi et al. 1997, Stokoe et al. GPCR pathways affecting the activity of AC are particu- 1997). Finally, PKB/Akt activates PDE3B, which degrades larly numerous, emphasizing the central role of the cAMP- cAMP to 50-AMP (Choi et al. 2006). This inactivates PKA dependent pathway in the regulation of TG hydrolysis. leading to reduced phosphorylation of HSL and PLIN1 and Thyroid-stimulating hormone (TSH) and the melano- suppression of lipolysis. cortins (MCs) adrenocorticotrophic hormone (ACTH) and Interestingly, in a study predating the elucidation of melanocyte-stimulating hormone stimulate AC by acti-

the canonical insulin signaling pathway, it was demon- vation of the Gs-coupled TSH receptor (Laugwitz et al. strated that besides the inhibitory effect on PKA-mediated 1996, Endo & Kobayashi 2012)andMCreceptors signaling, insulin also acts to reduce lipolysis in primary (Cho et al. 2005, Rodrigues et al. 2013) respectively (Fig. 3).

ANGPTL4 Growth hormone NPY-Y Glucocorticoids 1 A1-R HM74a MC ‘ANGPTL4-r’ α β 2-AR 1/2-AR GPR81 TSH-r

AC AC Gi Gs Gi Gs TNFR1 ATP ATP

cAMP

PKA IR Three FFA P P + glycerol HSL PLIN1 PLIN1 MGL ATGL PLIN1 DG MG CGI-58 Lipid droplet TG

Figure 3 Alternative signaling pathways in lipolysis. Black and red lines indicate pro- suppress lipolysis by inhibition of AC. Pro-inﬂammatory signaling through lipolytic and anti-lipolytic signaling events, respectively. Arrows indicate the TNF-a receptor (TNFR-1) increases lipolysis by suppressing antilipolytic

stimulation and blunt lines indicate inhibition. Dashed lines illustrate the signaling mediated by the insulin receptor (IR) and a2-adrenergic receptors

indirect lipolytic effects of growth hormone and glucocorticoids by (a2-ARs). For clarity, the intermediate intracellular steps in the different modulation of receptor sensitivities and ANGPTL4-mediated signaling. signaling pathways have been omitted. AC, adenylyl cyclase; ANGPTL4,

Although the identity of the ANGPTL4 receptor is unknown, the angiopoietin-like protein 4; ATGL, adipose triglyceride lipase; b1/2-ARs,

intracellular signaling has been shown to involve activation of AC. b1- and b2-adrenergic receptors; CGI-58, comparative gene

Stimulation of the Gs-protein-coupled melanocortin (MC) receptor and TSH identiﬁcation-58; DG, diacylglycerol; FFA, free fatty acid; Gi, inhibitory

receptor (TSH-r) also increases intracellular cAMP levels through activation G-protein; Gs, stimulating G-protein; HSL, hormone-sensitive lipase; MG,

of AC. Conversely, the Gi-protein-coupled receptors for NPY/PYY (NPY-Y1), monoacylglycerol; MGL, monoglyceride lipase; PKA, protein kinase A; adenosine (A1-R), b-hydroxybutyrate (HM74a), and lactate (GPR81) PLIN1, perilipin 1; TG, triglyceride; TSH, thyroid-stimulating hormone. Journal of Molecular Endocrinology

TSH-mediated lipolysis has been found to be particularly lipolysis has been underscored by elegant studies in important in neonates and newborns because physiologi- rodents demonstrating that isolated stimulation of the cal levels of TSH, as opposed to adrenaline or nor- hypothalamus with insulin suppresses peripheral AT adrenaline, potently stimulate human lipolysis at this lipolysis (Scherer et al. 2011). developmental stage (Marcus et al. 1988, Janson et al. The fasting-induced circulating factor angiopoietin- 1995). By contrast, although a strong lipolytic potential like protein 4 (ANGPTL4) is a well-established negative of the MCs has been observed in several animal species, regulator of lipid uptake in rodent adipocytes through including rodents, hamsters, guinea pigs, and rabbits inhibition of extracellular lipoprotein lipase activity (Richter & Schwandt 1983, Ng 1990), they seem to have (Koster et al. 2005, Sukonina et al. 2006, Lafferty et al. limited effects on human lipolysis (Bousquet-Melou et al. 2013). Recently, however, ANGPTL4 has also been found 1995, Kiwaki & Levine 2003). to be intimately involved in the regulation of intracellular Neuropeptide Y (NPY) and peptide YY (PYY) are cAMP-mediated lipolysis in mice (Gray et al. 2012). By an released from sympathetic neurons and inhibit AC by as yet unidentiﬁed mechanism, extracellular ANGPTL4

binding to the Gi-protein-coupled NPY-receptor (NPY-Y1; was shown to act independently of b-AR activation to Fig. 3) on human adipocytes (Serradeil-Le et al. 2000). Also, increase intracellular cAMP production via activation of in humans, the highest expression of NPY receptors has AC (Gray et al.2012). The identity of the putative been found in subcutaneous AT (Castan et al. 1993), ANGPTL4 receptor responsible for this effect in adipocytes suggesting that the impact of NPY/PYY on lipolysis is is unknown and is currently under investigation (Koliwad depot speciﬁc. The importance of neuronal regulation of et al. 2012).

In rat and human adipocytes, extracellular adenosine lipolytic effect of GH is counteracted by the AC inhibitor

efficiently inhibits lipolysis via the Gi-coupled adenosine acipimox (a niacin derivative; Nielsen et al. 2001, 2002). receptor (A1-R; Fig. 3; Lonnroth et al. 1989, Liang et al. Notably, stimulation with GH does not increase lipolysis 2002). However, in humans, the concentrations required in explants of human AT (Fain et al. 2008) or isolated in the interstitial fluid for a significant reduction of human adipocytes (Marcus et al. 1994), but the sensitivity lipolysis have been found to be at the very high end of toward b-adrenergic agonists is enhanced by the presence the physiological range and therefore of uncertain of GH in the culture medium (Marcus et al. 1994). In line significance (Lonnroth et al. 1989). The ketone body with this, it has recently been demonstrated that GH b-hydroxybutyrate (b-OHB) has been shown to inhibit administration in human subjects increases ANGPTL4

lipolysis in vitro by activating the human Gi-coupled levels in plasma (Clasen et al. 2013). Given the permissive receptor HM74a (Fig. 3; Taggart et al. 2005). HM74a, effect of this protein on cAMP-mediated lipolysis, it seems which is the ortholog of the mouse PUMA-G receptor, is likely that elevations in systemic ANGPTL4 levels could be also the target of the lipid-lowering drug nicotinic acid one of the mechanisms by which GH stimulates AT (niacin; Tunaru et al. 2003). Importantly, the observed lipolysis. Interestingly, the responsiveness toward GH inhibition of lipolysis by b-OHB was obtained at concen- stimulation varies among human AT depots, and visceral trations similar to those seen in humans during fasting, AT has been shown to be particularly sensitive to the suggesting a feedback mechanism by which b-OHB can lipolytic effects of GH (Nam et al. 2001, Pasarica et al. 2007, regulate its own production in order to prevent ketoaci- Plockinger & Reuter 2008). dosis during starvation (Taggart et al. 2005). Similarly, the receptor responsible for the antilipolytic effect of lactate Glucocorticoids has been identiﬁed as GPR81 (Fig. 3; Cai et al. 2008, Liu

et al. 2009a), which is a Gi-coupled receptor highly In a manner similar to GH, the lipolytic effects of homologous to HM74a (Cai et al. 2008). Like b-OHB, glucocorticoids have been attributed to an increased lactate inhibits lipolysis in adipocytes from several b-adrenergic responsiveness and a reduction of insulin- mammalian species including primates, rodents, and mediated antilipolysis (Fig. 3). Thus, in rat adipocytes, humans at concentrations within the normal physiologi- dexamethasone treatment has been shown to promote cal range (Liu et al. 2009a). Consequently, it has been PKA-mediated lipolysis by reducing the mRNA and protein hypothesized that GPR81 could act as a sensor of hypoxia expression levels of PDE3B (Xu et al. 2009). Also, by suppressing lipolysis in response to increased lactate dexamethasone potentiates the response toward b-AR

Journal of Molecular Endocrinology production (Cai et al. 2008). agonists both by inducing an increase in the number of b-ARs and by increasing the catalytic response of AC toward receptor-mediated activation (Lacasa et al. 1988). Growth hormone In agreement with this, elevated cortisol levels in humans Growth hormone (GH) potently and dose dependently have been found to reduce the postprandial suppression of stimulates lipolysis in humans (Hansen et al. 2002). In FFA release, suggesting a decreased antilipolytic effect of mice, knockout (KO) of the GH receptor renders the insulin (Dinneen et al. 1993). As for GH, glucocorticoid- animals susceptible to obesity, while GH over-expression mediated lipolysis in rodents is partially dependent on an results in a lean phenotype (Berryman et al. 2004, 2006). increase in ANGPTL4 (Koliwad et al. 2009, Gray et al. The nature of the molecular pathway involved in 2012), indicating that these hormones share some of GH-mediated lipolysis is not entirely clear. However, the mechanisms by which they stimulate lipolysis. results from animal studies indicate that it involves However, acute in vivo studies on humans have also remodeling of the cAMP-dependent regulatory signaling demonstrated additive independent effects of GH and pathways such that the responsiveness toward cortisol on lipolysis, suggesting that alternative, and b-adrenergic signaling is increased (Doris et al. 1994, distinct, lipolytic pathways exist for these hormones Yang et al. 2004) while insulin sensitivity is reduced (Djurhuus et al. 2004). (Chen et al. 2001, Johansen et al. 2003; Fig. 3). Although deﬁnitive mechanistic evidence is lacking, this model has Tumor necrosis factor a been supported by human studies. Thus, GH administration acutely stimulates lipolysis and causes peripheral Multiple effects of the pro-inﬂammatory cytokine tumor insulin resistance (Nellemann et al. 2013). Also, the in vivo necrosis factor-a (TNFa) on the lipolytic pathway have

been described. Acting through TNF receptor 1 ( Fig. 3)in Exercise adipocytes from mice (Sethi et al. 2000) and humans Physical exercise is the other major situation in which (Ryden et al. 2002), TNFa activates the three MAPKs lipolysis is stimulated in humans and this is believed to p42/44, JNK, and p38 of which p42/44 and JNK are involve the concerted action of several signaling pathways involved in the induction of lipolysis (Ryden et al. 2002). (Frayn 2010). Thus, circulating levels of adrenaline, In human fat cells, PDE3B protein expression is decreased noradrenaline, ANP, GH, and cortisol increase and insulin dramatically by TNFa (Zhang et al. 2002) and in rat decreases in proportion to exercise intensity, and these adipocytes antilipolytic signaling via the a2-AR is blunted gradual changes are reﬂected in the magnitude of the by TNFa by speciﬁc proteasomal degradation of Gi (Gasic resulting lipolytic response (Moro et al. 2007b). Further- et al. 1999, Botion et al. 2001). The combined effect of more, the adrenergic responsiveness of subcutaneous AT these alterations of the insulin and a2-AR signaling is altered with a shift from predominant a-adrenergic pathway is an increased intracellular cAMP level and a suppression during rest toward predominant b-adrenergic resulting activation of PKA-mediated lipolysis. In addition stimulation during exercise (Arner et al. 1990), and in the to modulation of antilipolytic signaling, exposure to TNFa post-exercise recovery phase, b-AR blockade has been increases ATGL activity due to remodeling of core shown to dramatically reduce plasma levels of FFA and components of the lipolytic machinery. This is discussed et al in the following section. glycerol (Wijnen . 1993). Similar to fasting conditions, GH and cortisol are likely to be some of the hormonal mediators of these exercise-induced alterations in adre- Physiological regulation of human AT lipolysis nergic responsiveness (Kanaley et al. 2004). The primary adrenergic stimulus of AT during exercise originates from As discussed earlier, the lipolytic rate in human AT is circulating catecholamines, with only a minor contri- determined by a delicate balance between several regulat- bution from noradrenaline released from sympathetic ory pathways. In healthy subjects, this regulation facili- neurons (Stallknecht et al. 2001, de Glisezinski et al. tates a proper lipolytic response to changes in systemic 2009). Additionally, the NPs have been found to play a nutrient demand. prominent role in exercise-induced lipolysis in humans (Moro et al. 2004b, de Glisezinski et al. 2009), and they Feeding/fasting have been suggested to account for most of the non- Following the ingestion of a meal, the post-prandial adrenergic lipolytic signaling in AT during exercise (Moro

Journal of Molecular Endocrinology increase in plasma insulin efficiently suppresses lipolysis et al. 2006, Lafontan et al. 2008). to promote the storage of dietary lipids (Roust & Jensen 1993, Jensen 1995). Conversely, in the fasting state, FFA Depot-specific regulation of lipolysis mobilization is promoted by the combined effects of reduced plasma insulin and increased release of adrenaline AT is not a homogenous organ, and significant regional and noradrenaline (Gjedsted et al. 2007). Also, it has been differences exist between depots in terms of hormonal demonstrated that lipolysis is further promoted by a responsiveness and metabolic activity. Also, the distribution combination of increased b-adrenergic sensitivity and of body fat is gender specific, with men generally having decreased insulin sensitivity in AT during fasting (Jensen a more central (upper-body) and women a more peripheral et al. 1987). Similarly, in obese individuals subjected to (lower-body) fat deposition (Demerath et al.2007). a hypocaloric diet (!3 MJ/day) for 28 days, lipolytic Regarding the lipolytic activity of the different depots, stimulation by b-adrenergic agonists as well as by ANP visceral and subcutaneous abdominal ATs are generally and BNP was enhanced significantly (Sengenes et al. 2002). more responsive toward lipolytic stimuli like catechol- These fasting-induced changes in hormonal sensitivity amines or prolonged fasting than subcutaneous gluteal are mediated, at least in part, by GH, which is elevated and femoral fat (Gjedsted et al. 2007, Manolopoulos et al. significantly during prolonged fasting (Norrelund et al. 2012). The reduced lipolytic effect of catecholamines in

2001, 2003, Vendelbo et al. 2010). Likewise, the diurnal lower-body fat depots is caused by enhanced a2-AR and ﬂuctuations in serum FFA levels mirror the pulsatile reduced b-AR responsiveness compared with upper-body secretion pattern of GH, and the nocturnal increase in depots (Manolopoulos et al. 2012). Additionally, in upper- FFA during sleep is virtually absent in GH-deﬁcient body obese women, the antilipolytic effect of insulin is patients (Jorgensen et al. 1990). blunted in the abdominal depots, which enhances

lipolysis further (Nellemann et al. 2012). Another import- groups (Jenkins et al. 2004, Villena et al. 2004, Zimmer- ant difference between upper- and lower-body subcu- mann et al. 2004). Initially named ATGL (Zimmermann taneous AT is the primary way by which adipogenesis et al. 2004), phospholipase A2x (Jenkins et al. 2004), and occurs as obesity develops. Thus, AT can expand either via desnutrin (Villena et al. 2004), the enzyme is now formally an increase in the number of fat cells (i.e. hyperplasia) or annotated as patatin-like phospholipase domain- by enlargement of the existing adipocytes (i.e. hypertro- containing protein 2 (Wilson et al. 2006). Expectedly, phy), of which the latter has been found to be an the expression of ATGL in mice has been found to be independent marker for increased metabolic risk (Weyer highest in white AT (WAT) and brown AT (BAT), but the et al. 2000, Lundgren et al. 2007). Importantly, irrespective transcript has been identified at lower levels in virtually all of gender, subcutaneous abdominal AT is more prone to tissues studied (Villena et al. 2004, Kershaw et al. 2006). expansion by hypertrophy than subcutaneous femoral AT, The transcriptional control of ATGL gene expression is which preferentially undergoes hyperplasia (Tchoukalova complex. A peroxisome proliferator-activated receptor g et al. 2010). (PPARg)-responsive element has been identified in the As a consequence of these regional differences in promoter sequence of the mouse Atgl gene (Kim et al. adipogenesis and lipolytic responsiveness, it has been 2006), and accordingly thiazolidinediones (PPARg ago- found repeatedly that upper-body obesity, but not lower- nists) like rosiglitazone increase Atgl expression (Kim et al. body obesity, is associated with elevated systemic FFA 2006, Liu et al. 2009b). Furthermore, Atgl mRNA levels and metabolic dysfunction (Nielsen et al. 2004, expression in 3T3-L1 adipocytes is negatively regulated Piche et al. 2008, Lapointe et al. 2009, Amati et al. 2012). by insulin as well as by TNFa-mediated p42/44 MAPK In fact, it has been suggested that the preference of activation (Kim et al. 2006). Several additional studies gluteofemoral fat for ‘trapping’ lipids serves as a ‘metabolic have addressed the regulation of ATGL expression, and sink’ providing metabolic and cardiovascular protection it has been found that in humans, ATGL protein is from the deleterious effects of an excessive daily influx of upregulated by fasting (Nielsen et al. 2011), while in dietary lipids (Manolopoulos et al. 2010). The regulation mice, the mRNA is suppressed by feeding (Kershaw et al. and implications of these gender- and depot-specific 2006), but upregulated by glucocorticoids (Villena et al. differences in terms of AT metabolism and signaling has 2004), and by SIRT1-mediated activation of the transcrip- been covered in great detail in a recent review (White & tion factor Foxo1 (Chakrabarti et al. 2011, Shan et al. 2013). Tchoukalova 2014). However, numerous studies have found that changes in mammalian ATGL mRNA and protein levels are often Journal of Molecular Endocrinology The lipolytic pathway: enzymes and reciprocal, suggesting that ATGL is subject to extensive post-transcriptional regulation (Steinberg et al. 2007, Li co-regulators et al. 2010, Nielsen et al. 2011, 2012). The core enzymatic machinery for TG hydrolysis in AT ATGL is a specific TG hydrolase, and the activity consists of ATGL and HSL. Studies of ATGL-KO mice have toward other lipid substrates like DG, MG, retinylesters revealed that the absence of ATGL reduces the lipolytic (RE), or cholesterylesters (CE) is very limited (Zimmermann response of adipocytes to b-AR stimulation by w70%, and et al.2004). Although the 3D structure of ATGL has not by adding a specific HSL inhibitor lipolysis is reduced by been reported, studies on mutated and truncated human more than 95% (Schweiger et al. 2006). Furthermore, both and murine ATGL have revealed that the N-terminal half the basal and stimulated lipolytic capacity of human and of the enzyme contains the catalytic patatin domain mouse adipocytes are increased by ATGL overexpression (Duncan et al. 2010), while the C-terminal part is believed and deceased by ATGL silencing (Kershaw et al. 2006, to be involved in regulation of enzymatic activity and to Bezaire et al. 2009). By contrast, HSL overexpression or mediate the interaction between ATGL and LDs silencing does not affect the basal lipolytic rates in human (Kobayashi et al. 2008, Schweiger et al. 2008). The two adipocytes, but the maximal stimulated lipolytic rate is serine residues Ser404 and Ser428 in the C-terminal part of decreased by reduced HSL levels (Bezaire et al. 2009). the human ATGL sequence (corresponding to Ser406 and Ser430 in murine ATGL) have been identified as phosphorylation sites (Bartz et al. 2007). However, the role of Adipose TG lipase these sites in the regulation of ATGL activity, and the The important function of ATGL as a TG hydrolase was identity of the upstream kinases is somewhat unclear. discovered simultaneously in 2004 by three different Thus, Ser406 has been suggested to be a consensus site for

AMP-activated protein kinase (AMPK), and in murine 3T3- L1 adipocytes, it was shown that pharmacological stimulation of lipolysis with the AMPK agonist AICAR was CGI-58 406 ATGL PLIN1 dependent on ATGL Ser phosphorylation (Ahmadian A CGI-58 ATGL G0S2 et al. 2011). However, another study found that phos- TG DG phorylation of Ser404 in human ATGL was increased by PLIN1 b-adrenergic stimulation, while AICAR treatment had no Lipid droplet effect (Pagnon et al. 2012). Additionally, in mouse AT, Acute stimulation Ser406 phosphorylation was increased with fasting, exer- PKA cise, and ex vivo stimulation of the cAMP-dependent B pathway in a PKA-dependent manner, thereby implicating this kinase in the phosphorylation of ATGL (Pagnon et al. P P P 404 ATGL PLIN1 2012). Notably, in human skeletal muscle (SM), Ser P CGI-58 CGI-58 phosphorylation is also associated with PKA signaling and P ATGL TG DG G0S2 not with AMPK signaling, indicating that PKA is indeed the P PLIN1 upstream kinase for this site (Mason et al. 2012). Whether PKA and/or AMPK are responsible for phosphorylation of Prolonged stimulation Ser430 is currently unknown, and so far no reports have been published on the functional role of this site. Degradation P G0S2 P C ATGL PLIN1 P P CGI-58 ATGL: activation by CGI-58 CGI-58 P ATGL TG DG TG The primary way by which ATGL activity is increased P PLIN1 under acute lipolytic stimulation is via interaction with DG the co-activator CGI-58 (also known as a/b hydrolase

domain-containing protein 5; Fig. 4A and B; Lass et al. Figure 4 2006). Activation depends on the interaction of CGI-58 Regulation of ATGL. (A) In the basal state, CGI-58 is complexed with PLIN1 with the patatin domain of ATGL (Schweiger et al. 2008, and ATGL activity is low. (B) Upon phosphorylation of PLIN1, CGI-58 is released and associates with ATGL, which increases ATGL activity. In this Cornaciu et al. 2011) and requires direct protein–protein phase, a fraction of the ATGL pool is dominantly inhibited by G0S2. (C) If

Journal of Molecular Endocrinology interactions between ATGL and CGI-58 (Granneman et al. the lipolytic stimulation persists, gradual degradation of G0S2 promotes a 2007, Cornaciu et al. 2011). Also, mutational studies have further increase in ATGL activity. TG, triglyceride; ATGL, adipose TG lipase; CGI-58, comparative gene identification-58; DG, diacylglycerol; G0S2, G0/G1 shown that additional binding of CGI-58 to the LD is switch gene 2; PKA, protein kinase A; PLIN1, perilipin 1. crucial in order to activate ATGL (Gruber et al. 2010). The molecular mechanism by which CGI-58 activates ATGL Consequently, as downstream inflammatory stress kinases is unclear, but it could potentially involve induction can impair insulin signaling, CGI-58 seems to be involved of conformational changes, presentation of substrate, or in cross talk between insulin sensitivity and inflammation, removal of reaction products. Interestingly, in addition to at least in mice. its role in regulating lipolysis, both mouse and human CGI-58 has been identified as a CoA-dependent lysopho- ATGL: inhibition by G0S2 sphatidic acid acyltransferase (LPAAT; Ghosh et al. 2008, Gruber et al. 2010, Montero-Moran et al. 2010), and it has Recently, the protein product of G0/G1 switch gene 2 been speculated that it promotes lipolysis by channeling (G0S2) was identified as an inhibitor of ATGL (Yang et al. fatty acids released from TG hydrolysis into phospholipids 2010). In mice, G0S2 is primarily expressed in brown to reduce end product inhibition of ATGL and HSL and white adipocytes, but a significant expression has (Montero-Moran et al. 2010). Although the potential also been detected in liver, heart, and SM (Zandbergen relevance of this LPAAT activity of CGI-58 in lipolytic et al. 2005). Murine G0S2 mRNA and protein expression regulation remains to be investigated, CGI-58-derived has been shown to be induced by insulin and PPARg phospholipids are essential second messengers in murine and suppressed by lipolytic agents like TNFa and liver and AT when exposed to pro-inflammatory cytokines the b-AR agonist isoprenaline (Zandbergen et al. 2005, like TNFa, interleukin 1b (IL1b), and IL6 (Lord et al. 2012). Yang et al. 2010). Like CGI-58, G0S2 interacts directly with

the catalytic patatin domain of ATGL (Yang et al. 2010, et al. 2009, Schoiswohl et al. 2010). Without sufficient fuel Cornaciu et al. 2011), but the ATGL–G0S2 interaction is from lipid substrates, they rely primarily on carbohydrate independent of the ATGL–CGI-58 interaction, and inhi- metabolism for energy conversion resulting in rapid bition by G0S2 appears to be dominant to activation by depletion of hepatic and SM glycogen stores (Huijsman CGI-58 (Fig. 4B; Yang et al. 2010, 2011, Schweiger et al. et al. 2009, Schoiswohl et al. 2010). Consequently, when 2012). Human G0S2, like the murine ortholog, inhibits subjected to moderate exercise or short-term fasting, the ATGL in a dose-dependent manner and is also involved mice become hypoglycemic, and if fasting is extended in regulating the intracellular localization of ATGL by beyond a modest 8–12 h, they develop signs of severe recruiting it to LDs (Schweiger et al. 2012). energy starvation like hypothermia, lethargy, reduced It has been proposed that G0S2 acts as a long-term oxygen consumption, and loss of lean mass (Haemmerle regulator of lipolysis, as G0S2 protein levels are gradually et al. 2006, Schoiswohl et al. 2010, Wu et al. 2012). reduced during prolonged lipolytic stimulation resulting in Similarly, in spite of massively increased BAT mass, ATGL- increased ATGL activity (Fig. 4C; Yang et al.2010). Notably, KO mice are unable to increase thermogenesis in response G0S2 protein and mRNA expression is dramatically reduced to cold exposure, indicating that the mobilization of lipid in human AT by prolonged physiological stimulation of fuel in BAT is defective (Haemmerle et al. 2006). In lipolysis with a 72-h fast (Nielsen et al.2011) and similar addition to the abnormal substrate metabolism, ATGL effects have been observed in birds (Oh et al.2011) and pigs deficiency causes pancreatic steatosis leading to impaired (Ahn et al.2013). However, it is currently not known if the insulin secretion and hypoinsulinemia (Peyot et al. 2009). association between ATGL and G0S2 is dynamic and subject Interestingly, however, despite the massive ectopic lipid to regulation or if G0S2-mediated ATGL inhibition accumulation and b-cell dysfunction, the ATGL-deficient primarily depends on the intracellular levels of G0S2. mice have improved whole-body insulin sensitivity and Recent evidence has suggested the latter although. Thus, glucose tolerance compared with WT animals (Haemmerle in 3T3-L1 adipocytes, stimulation with TNFa for up to 16 h et al. 2006, Peyot et al. 2009). Specifically, muscle and WAT reduces G0S2 levels gradually, while the rate of lipolysis insulin signaling is improved, although in BAT and liver it increases nearly proportionally to the G0S2 reduction (Yang is reduced (Kienesberger et al. 2009). et al.2011). Conversely, overexpression of G0S2 signi- The key role of defective TG catabolism in the ficantly reduces the TNFa mediated lipolytic response. phenotype of ATGL-KO mice has recently been supported Murine G0S2 is a short-lived protein with a half-life of with the generation of transgenic mice with AT-specific !1 h, and its stability can be greatly improved by inhibition overexpression of G0S2 (Heckmann et al. 2014). Like

Journal of Molecular Endocrinology of the proteasomal pathway (Yang et al.2011). Hence, it ATGL-deficient mice, WAT and BAT mass is increased in appears that one of the mechanisms by which TNFa these animals due to impaired basal and stimulated promotes adipocyte lipolysis is by suppressing G0S2 lipolysis, but glucose and insulin tolerance is improved. mRNA expression leading to cytosolic depletion of G0S2 Moreover, thermogenesis is attenuated leading to defec- protein through proteasomal degradation and, conse- tive cold adaptation, and the fasting-induced switch from quently, increased ATGL activity. carbohydrate to fatty acid metabolism is severely impaired (Heckmann et al. 2014). Conversely, mice with global G0S2 KO are lean and resistant to high-fat diet-induced Animal models of ATGL deficiency obesity and hepatic steatosis (Zhang et al. 2013). Further- Insight into the crucial role of ATGL in whole-body TG more, hepatic fatty acid metabolism is enhanced as G0S2 metabolism has been provided from the characterization ablation accelerates ketogenesis and gluconeogenesis of ATGL-deficient mice (Haemmerle et al. 2006). These while glycogen breakdown is impaired (Zhang et al. animals exhibit massive ectopic lipid accumulation in 2013). Combined with the observations from Atgl-KO virtually all tissues and especially in AT, liver, SM, and mice, these results support a defining role for ATGL- heart (Haemmerle et al. 2006). Accordingly, the animals mediated lipolysis in whole-body substrate partitioning become obese even on a low-fat diet, and they suffer from and metabolism, at least in mice. premature death due to severe cardiac steatosis and dysfunction (Haemmerle et al. 2006, Schrammel et al. ATGL in human obesity and metabolic disease 2013). Furthermore, the normal fasting- or exercise- induced rise in plasma FFA is absent in ATGL-KO animals The available literature on the expression patterns of ATGL indicating a failure to increase lipolysis in WAT (Huijsman in human obesity is somewhat conflicting. In a study

among lean and obese women, no difference in ATGL glucose challenge (Natali et al. 2013), while insulin protein levels were found in subcutaneous abdominal AT resistance is more common in patients with severe (Ryden et al. 2007). Conversely, in mixed populations of muscular involvement and hepatic steatosis (Laforet men and women, ATGL mRNA was increased in subcu- et al. 2013). The clinical manifestations of loss-of-function taneous abdominal AT in obesity, but the protein levels mutations in CGI-58 are similar to those observed in were reduced (Steinberg et al. 2007, Yao-Borengasser et al. functional ATGL deficiency except that these patients do 2011) and a negative correlation was found between BMI not develop myopathy (Igal et al. 1997). Instead, they and ATGL protein expression (Yao-Borengasser et al. 2011). suffer from severe ichthyosis (Chanarin et al. 1975, Lefevre Furthermore, in paired biopsies, ATGL mRNA expression et al. 2001) and, accordingly, the two types of NLSD are was lower in visceral than in subcutaneous abdominal AT known as NLSD with myopathy (NLSDM) and NLSD with (Yao-Borengasser et al. 2011), and when comparing visceral ichthyosis (NLSDI, also known as Chanarin–Dorfman AT from obese and lean subjects, ATGL mRNA was syndrome) (Fischer et al. 2007). The epidermal defects increased in obesity (Steinberg et al. 2007, Tinahones observed in NLSDI are not present in NLSDM, suggesting et al. 2010), while the protein levels were unaffected an ATGL-independent function of CGI-58, possibly as a (Steinberg et al. 2007). Similarly, in a recent comparison LPAAT in the synthesis pathway of glycerophospholipids of lean and obese men, only the mRNA of ATGL was and acylceramides required for the formation and increased in visceral fat in obesity, but in subcutaneous maintenance of the skin permeability barrier (Igal & abdominal fat, ATGL protein was increased in the obese Coleman 1996, Radner et al. 2009). Consistently, KO of subjects (De Naeyer et al. 2011). Furthermore, among obese CGI-58 in mice results in a neonatal lethal phenotype males and females with either normal or impaired insulin caused by leaky skin, and the pups die from desiccation sensitivity, insulin resistance has been shown to be within hours after birth (Radner et al. 2009). An overview associated with reduced ATGL protein and mRNA in of studies on genetic deficiencies affecting ATGL function subcutaneous abdominal AT (Jocken et al. 2007) and in humans and animal models is listed in Table 1. reduced ATGL mRNA in visceral AT (Berndt et al. 2008). However, whereas the available data on ATGL expression Hormone-sensitive lipase in obesity is inconclusive, the expression of CGI-58 seems to be remarkably stable between depots (Yao-Borengasser HSL was discovered in rat AT in the early 1960s as a et al. 2011) and in obesity (Steinberg et al. 2007). lipolytic enzyme, which was inducible by fasting Gender-specific differences may explain some of the and stimulation with ACTH or adrenaline and inhibited

Journal of Molecular Endocrinology discrepancies between the studies on ATGL, but in light of by insulin (Hollenberg et al. 1961, Rizack 1964, Vaughan the substantial body of conflicting data, the role of human et al. 1964). ATGL in the pathogenesis of metabolic disease in obesity is Like ATGL, HSL is expressed in most tissues examined, currently unclear. with the highest expression found in WAT and BAT By contrast, defective ATGL-mediated lipolysis has (Kraemer et al. 1993). The mRNA is generated from a been unequivocally identified as the primary defect in the single gene controlled by a number of alternative inherited monogenic disorder neutral lipid storage disease promoters that produce several different tissue-specific (NLSD; Lefevre et al. 2001, Lass et al. 2006, Fischer et al. isoforms of the HSL protein that range in size from 2007). Patients with loss-of-function mutations affecting w85 kDa and up to 130 kDa (Langin et al. 1993, Mairal ATGL are characterized by ectopic TG accumulation and et al. 2002). The HSL isoform found in human AT is a visceral obesity, skeletal and cardiac myopathy, and 786 aa protein with an apparent molecular weight of variable degrees of hepatic and pancreatic steatosis w88 kDa (Langin et al. 1993). (Schweiger et al. 2009, Laforet et al. 2013, Natali et al. Efficient lipid hydrolysis by HSL requires the lipase to 2013).However,themetabolicphenotypeofthese form a complex with cytosolic fatty acid-binding protein 4 patients is heterogeneous. Thus, some are insulin resistant (FABP4), which acts as a molecular chaperone by shuttling and develop type 2 diabetes (Laforet et al. 2013), while the FFA generated by HSL out of the cell (Fig. 5A; others have normal insulin sensitivity but impaired Furuhashi & Hotamisligil 2008). Upon stimulation of glucose tolerance (Natali et al. 2013). This probably reflects lipolysis, HSL and FABP4 associate in the cytosol and the individual differences in the pattern of ectopic lipid complex translocate to LDs (Jenkins-Kruchten et al. 2003, deposition: patients with extensive pancreatic steatosis Smith et al. 2007). Consistently, in FABP4-KO mice, the generally have an impaired insulin response to an oral lipolytic capacity is reduced, and the intracellular FFA

Table 1 Overview of genetic studies on ATGL and CGI-58 function

Diagnosis/genetic Affected Species model protein Highlights of the study Reference

Human NLSDI CGI-58 Case report: clinical manifestations and proposal Igal et al. (1997) of diagnostic criteria Human NLSDI CGI-58 Identification of defects in CGI-58 as causative for Lefevre et al. (2001) NLSDI Human NLSDI CGI-58 Activation of ATGL and rescue of NLSDI by CGI-58 Lass et al. (2006) Human NLSDM ATGL Case report: identification of truncations in Fischer et al. (2007) human ATGL Human NLSDM ATGL Identification of biochemical defects in truncated Kobayashi et al. (2008) ATGL Human NLSDM ATGL Case report: magnetic resonance imaging of Laforet et al. (2013) muscles and metabolic characterization Human NLSDM ATGL Case report: body composition and glucose/lipid Natali et al. (2013) metabolism Mouse KO ATGL and Measurement of lipolysis in AT explants from KO Schweiger et al. (2006) HSL animals Mouse KO ATGL Phenotyping of KO animals Haemmerle et al. (2006) Mouse KO ATGL Insulin sensitivity and glucose/lipid metabolism Kienesberger et al. (2009) Mouse KO ATGL and Substrate partitioning and metabolism during rest Huijsman et al. (2009) HSL and exercise Mouse KO ATGL Evaluation of effects on insulin secretion Peyot et al. (2009) Mouse KO CGI-58 Phenotyping of KO animals Radner et al. (2009) Mouse KO with cardio-specific ATGL Lipid/glucose metabolism during exercise Schoiswohl et al. (2010) expression Mouse AT-specific KO ATGL Phenotyping and fed/fasting metabolism Wu et al. (2012) Mouse KO with cardio-specific ATGL Cardiac metabolism Schrammel et al. (2013) expression Mouse KO G0S2 Phenotyping of transgenic animals Zhang et al. (2013) Mouse AT-specific overexpression G0S2 Phenotyping of transgenic animals Heckmann et al. (2014)

levels in adipocytes are increased (Coe et al. 1999). Animal models of HSL deficiency Activation of HSL requires phosphorylation by PKA or The key role of HSL as a DG hydrolase in vivo was revealed Journal of Molecular Endocrinology PKG on specific regulatory serine residues. In rat HSL, with the generation of HSL-KO mice, which were found to these sites are Ser563, Ser659, and Ser660 (Stralfors et al. 1984, accumulate intracellular DG in AT, SM, cardiac muscle, and Garton et al. 1988, Anthonsen et al. 1998) corresponding testis (Haemmerle et al.2002). However, in contrast to to the human residues Ser552, Ser649, and Ser650 (Contreras ATGL-KO mice, HSL-deficient mice do not suffer from et al. 1998, Watt et al. 2006). The functional role of these 563 severe systemic lipid accumulation, although they tend to sites is different; phosphorylation of Ser is thought to promote the translocation of HSL from the cytosol to LDs have enlargement of internal organs like liver, heart, (Daval et al. 2005), while phosphorylation of Ser659 and pancreas, and spleen (Harada et al.2003). Surprisingly, the Ser660 is critical for activation of the intrinsic enzymatic WAT mass is slightly reduced, and they are resistant to high- activity (Anthonsen et al. 1998). Conversely, phosphoryl- fat diet-induced obesity and peripheral insulin resistance ation of rat HSL on Ser565 (human Ser554) by AMPK inhibits (Osuga et al.2000, Harada et al.2003, Park et al.2005). This HSL activation, most likely by steric hindrance of unexpected observation was found to be caused by a phosphorylation of the adjacent Ser563, thus preventing compensatory reduction in fatty acid esterification and the translocation of HSL to the LDs (Daval et al. 2005) de novo lipogenesis to counteract the reduced release of FFA (Fig. 5B). In terms of specificity, HSL is a much more to the circulation (Zimmermann et al.2003). However, promiscuous lipase than ATGL and readily hydrolyze similar to ATGL-KO mice, HSL-KO mice have increased BAT several lipid substrates, including TG, DG, MG, RE, and mass and enlargement of brown adipocytes (Harada et al. CE in vitro (Fredrikson et al. 1981, Wei et al. 1997). Among 2003), but this is not associated with impaired thermo- the substrates and intermediates in TG lipolysis, the genesis, as they retain a normal sensitivity to cold exposure affinity for DG is approximately tenfold higher than for (Osuga et al.2000). An overview of animal studies on TG and MG (Fredrikson et al. 1981, 1986). genetic deficiencies affecting HSL is listed in Table 2.

FFA AB

cAMP FFA- FABP4 PDE3B 650 649 552 PKA 554 Ser Ser Ser ′ P P 5 -AMP Ser P P P P PKA HSL FABP4 AMPK HSL

552 P Ser P P P P P P P P P HSL FABP4 P HSL P P P P PLIN1 P PLIN1 P PLIN1 P PLIN1 DG MG DG MG Lipid droplet Lipid droplet

Figure 5 Regulation of HSL. (A) Phosphorylation of Ser552,Ser649, and Ser650 on requires cAMP-dependent phosphorylation on Ser552 by PKA. Conversely, human HSL promotes lipase activation and association with FABP4 in the AMPK is activated by 50-AMP and phosphorylate HSL on the adjacent cytosol. Subsequent translocation of this complex to the LD surface is Ser554. As phosphorylation on Ser552 and Ser554 is mutually exclusive, AMPK dependent on both HSL and PLIN1 phosphorylation and results in full reduces the LD association of HSL. AMPK, AMP-activated protein kinase; activation of HSL activity. Acting as a molecular chaperone, FABP4 shuttles DG, diacylglycerol; FABP4, fatty acid-binding protein 4; FFA, free fatty acid; the FFAs released by HSL from the LD to the plasma membrane of the HSL, hormone-sensitive lipase; MG, monoacylglycerol; PDE3B, phosphodi- adipocyte where they are secreted. (B) LD-targeting of cytoplasmic HSL esterase 3B; PKA, protein kinase A.

HSL in human obesity and metabolic disease Monoglyceride lipase

While changes in ATGL expression patterns in obesity MGL was identified in rats as an MG-specific lipase with no are of uncertain significance, the importance of HSL affinity toward TG or DG (Tornqvist & Belfrage 1976). The expression is somewhat more well established, although first MGL-KO mouse model has recently been generated

Journal of Molecular Endocrinology discrepancies certainly exist. Thus, the expression of HSL (Table 2), and these animals accumulate MG in WAT, mRNA in subcutaneous abdominal AT in obesity has been brain, and liver (Taschler et al. 2011). Also, it was found reported to be increased (Ray et al. 2009), reduced (Large that HSL partially compensated for the absence of MGL et al. 1999, Mairal et al. 2006), or not affected (Steinberg in AT, as the stimulated glycerol release was reduced by et al. 2007, De Naeyer et al. 2011). However, irrespective of a modest 43% compared with WT mice. However, upon gender, the majority of studies have found the correspond- specific inhibition of HSL in cultured fat pads, the ing HSL protein levels to be reduced in obesity (Large et al. MG-hydrolase activity was almost completely abolished 1999, Langin et al. 2005, Ryden et al. 2007, Ray et al. 2009). (Taschler et al. 2011). To date, no reports have been Similarly, in the obese state, insulin resistance is associated published indicating that MGL expression and enzymatic with a reduction in HSL mRNA and protein in subcu- activity in AT is regulated by hormonal signals or taneous abdominal AT (Jocken et al. 2007). In visceral AT, nutritional status, suggesting that the enzyme is constitu- HSL mRNA levels have consistently been found to be tively active in the lipolytic cascade. upregulated in obesity (Mairal et al. 2006, Steinberg et al. 2007, Ray et al. 2009, De Naeyer et al. 2011), but the LD-associated proteins: the CIDE family protein levels seem to be unaffected (De Naeyer et al. 2011) or possibly reduced (Ray et al. 2009). The PLIN proteins and the CIDE proteins constitute the So far, no human examples of loss-of-function two major families of LD-associated proteins in adipo- mutations affecting HSL have been reported, and in light cytes. The pro-apoptotic CIDE proteins (cell-death indu- of the relatively mild phenotype of HSL-KO mice, it seems cing DNA fragmentation factor-a-like effector) comprise unlikely that HSL deficiency per se is associated with severe three members: CIDEA, CIDEB, and CIDEC (also known as metabolic defects in humans. fat-specific protein of 27 kDa, Fsp27) (Yonezawa et al. 2011).

Table 2 Overview of genetic studies on HSL, MGL, and FABP4 functional relationship, they are now annotated as PLIN1 function (perilipin A), PLIN2 (adipophilin), PLIN3 (TIP-47), PLIN4 (S3-12), and PLIN5 (MLDP/OXPAT) (Kimmel et al. 2010). Genetic Affected Highlights Of the PLIN proteins, PLIN1 is particularly important in Species model protein of the study Reference the regulation of AT lipolysis, while the other family Mouse KO FABP4 Phenotyping of Coe et al. members are involved in adipogenesis and LD formation KO animals (1999) Mouse KO HSL Phenotyping of Osuga et al. (PLIN2), LD biosynthesis and stabilization (PLIN3), KO animals (2000) LD maturation (PLIN4) and regulation of lipolysis in Mouse KO HSL Involvement of Haemmerle oxidative tissues not expressing PLIN1 (PLIN5) (Bickel HSL in et al. (2002) whole-body et al. 2009). DG catabolism Multiple regulatory roles of murine PLIN1 have been Mouse KO HSL Lipid metab- Harada et al. described in the lipolytic cascade, and it has been olism in (2003) diet-induced suggested that PLIN1 is the ‘master regulator’ of PKA- obesity stimulated lipolysis in mice (Miyoshi et al. 2007). As such, Mouse KO HSL AT adaptations Zimmermann PLIN1 either directly or indirectly regulates the activity of to HSL et al. (2003) deficiency ATGL and HSL as well as their access to lipid substrates in Mouse KO HSL Insulin sensi- Park et al. the LDs. The LD targeting of HSL is partly governed by tivity and (2005) PLIN1; in the basal state, as much as half of the total glucose/lipid metabolism cellular HSL content is located in the cytoplasm, but PKA- Mouse KO MGL Phenotyping of Taschler et al. mediated PLIN1 and HSL phosphorylation has been KO animals (2011) shown to significantly enhance the co-localization and association of the two proteins on LDs (Miyoshi et al. CIDEA and CIDEC from mice and humans have recently 2006). However, studies on mutant PLIN1 lacking all been shown to be negative regulators of lipolysis (Nord- phosphosites have revealed that in the absence of strom et al. 2005, Puri et al. 2008, Christianson et al. 2010), phosphorylation of PLIN1, the PKA-mediated increase in HSL activity is blunted, although the lipase is phosphory- although their specific role is not well characterized yet. lated and translocated (Miyoshi et al. 2006). In other However, it appears that they promote lipid storage words, the lipolytic action of LD-associated and phos- through their involvement in LD formation, fusion, and phorylated HSL are critically dependent on PLIN1 phos- stabilization (Puri et al. 2008, Christianson et al. 2010, Ito Journal of Molecular Endocrinology phorylation in mouse adipocytes. et al. 2010). Accordingly, human CIDEC has been found Similarly, the activation of murine ATGL depends to interact with PLIN1 (see below) and the interaction on phosphorylation of PLIN1, although by a different between these two proteins is critical for the formation of mechanism. In the basal state, unphosphorylated PLIN1 large LD’s and unilocular adipocytes (i.e. cells containing a has been shown to negatively regulate ATGL by efficiently single big LD) (Grahn et al. 2013, Sun et al. 2013). The sequestering CGI-58, thereby preventing activation of mechanism by which the CIDE proteins inhibit lipolysis is ATGL (Granneman et al. 2007, 2009). Upon stimulation incompletely understood but it seems to involve shielding of lipolysis, phosphorylation of PLIN1 causes the dissoci- of the LDs from the action of lipases by providing a ation of CGI-58, which can then bind and activate ATGL physical barrier around the lipid core (Christianson et al. (Granneman et al. 2009; Fig. 4). Interestingly, a single 2010, Yang et al. 2013). amino acid in PLIN1 has been identified as the crucial residue for regulation of HSL- and ATGL-mediated 517 LD-associated proteins: the PLIN family lipolysis, as mutation of Ser in the mouse sequence almost completely abolishes PKA-stimulated FFA and The other family of LD-associated proteins has been glycerol release (Miyoshi et al. 2007). studied in much more detail. The PLIN proteins were originally called the PAT family after perilipin A, adipo- Animal models of deficiencies in LD-associated proteins philin, and tail-interacting protein of 47 kDa (TIP-47), and the family also includes the proteins S3-12 and myocyte In mice, the CIDE proteins exhibit a distinct expression LD protein (MLDP, also known as OXPAT) (Bickel et al. pattern. Thus, while CIDEA is predominantly expressed in 2009). However, due to their evolutionary, structural, and BAT (Zhou et al. 2003), CIDEB is almost exclusively

expressed in the liver (Li et al. 2007), and CIDEC is 2001). In cultured cells, overexpression of PLIN2 has primarily found in WAT (Nishino et al. 2008), suggesting a been shown to promote lipid storage by negatively tissue-specific role of these proteins. Consistently, CIDEA- regulating the access of ATGL to LDs (Listenberger et al. deficient mice are characterized by elevations in body 2007), but the underlying mechanism is currently temperature and overall metabolic rate due to accelerated unknown, and it remains to be determined whether BAT lipolysis and thermogenesis (Zhou et al.2003). PLIN1 deficiency is associated with increased PLIN2 Consequently, these mice are lean and resistant to diet- expression in humans in vivo. induce obesity and glucose intolerance. A liver-specific function of CIDEA has also been demonstrated, as hepatic LD-associated proteins in human obesity and CIDEA knockdown in genetically obese ob/ob mice reduces metabolic disease hepatic TG accumulation and LD size and accelerates overall energy expenditure (Zhou et al. 2012). Similarly, KO As for ATGL and HSL, the available data on the association of CIDEB results in lean mice with improved glucose between obesity and the expression of LD-associated tolerance, insulin sensitivity, and resistance to hepatic proteins are rather inconclusive. In the obese state, steatosis, but BAT metabolism is normal in these animals PLIN1 mRNA expression has been reported to be increased (Li et al. 2007). Instead, ketogenesis and overall metabolic in visceral AT (Ray et al. 2009, Tinahones et al. 2010) but rate is accelerated, suggesting an overall shift in substrate unaffected in subcutaneous abdominal AT (Ray et al. utilization toward lipid metabolism (Li et al. 2007). In 2009). However, others have found a negative correlation terms of metabolic rate, susceptibility to obesity, hepatic between BMI and visceral PLIN1 mRNA levels, suggesting steatosis, and disturbances in glucose homeostasis and that the expression is reduced in obesity (Moreno- metabolism, the overall phenotype of CIDEC-KO mice is Navarrete et al. 2013). In obesity, PLIN1 protein expression very similar to the phenotype of CIDEA- and CIDEB- is reduced in both visceral and subcutaneous abdominal deficient animals (Nishino et al. 2008, Toh et al. 2008). AT (Ray et al. 2009), and among obese subjects, the Notably, however, while the metabolic rate in BAT is protein levels in subcutaneous abdominal AT are lower in reduced, mitochondrial biogenesis, oxygen consumption, insulin-resistant subjects than in insulin-sensitive subjects and basal lipolytic rates are increased in WAT (Nishino (Moreno-Navarrete et al. 2013). et al. 2008). Consistently, WAT expression of specific Recently, two different loss-of-function mutations in factors inhibiting BAT differentiation is reduced and a PLIN1 have been identified in three patients diagnosed concomitant increase in the expression of BAT-specific with a rare autosomal dominant partial lipodystrophy

Journal of Molecular Endocrinology genes (e.g. UCP1) causes a shift toward a more brown-like (Gandotra et al. 2011a). Both mutations introduced a phenotype (Toh et al. 2008). Thus, in spite of tissue-specific frameshift causing the loss of three regulatory PKA sites, differences in the expression and regulation of the CIDE including the crucial Ser517. Consequently, these mutants proteins, they seem to share a crucial role in the regulation fail to sequester CGI-58 leading to permanently elevated of overall substrate partitioning and metabolism. basal lipolysis due to constitutive activation of ATGL In agreement with the role of PLIN1 as a negative (Gandotra et al. 2011b). The clinical manifestations of regulator of ATGL activity, PLIN1-deficient mice have these mutations included dyslipidemia, hypertension, been found to have dramatically decreased fat mass and lipoatrophy, hepatic steatosis, severe insulin resistance, increased basal lipolysis (Martinez-Botas et al. 2000, and type 2 diabetes. Remarkably, a very similar phenotype Tansey et al. 2001). This observation has been supported has been found in a patient carrying a loss-of-function by in vitro studies; stimulation of 3T3-L1 cells with TNFa mutation in the LD-targeting domain of CIDEC, was found to increase basal lipolysis due to a reduction in suggesting that the integrity of the interaction between PLIN1 protein levels, and this effect could be reversed by CIDEC and PLIN1 is crucial for proper LD dynamics and simultaneous overexpression of PLIN1 (Souza et al. 1998). lipolytic control in humans in vivo (Rubio-Cabezas et al. Furthermore, PLIN1-KO animals are lean and resistant to 2009). Additionally, the CIDEC-deficient AT had an diet-induced obesity, but nevertheless they are prone unusually high occurrence of multilocular adipocytes to develop glucose intolerance and peripheral insulin supporting a role for CIDEC in the formation and resistance (Martinez-Botas et al. 2000, Tansey et al. 2001). stabilization of large LD’s (Rubio-Cabezas et al. 2009). An Interestingly, in the absence of PLIN1, the expression of overview of studies on genetic deficiencies affecting PLIN2 is increased and, consequently, PLIN2 is the major LD-associated proteins in humans and animal models is LD-associated protein in these animals (Tansey et al. listed in Table 3.

Table 3 Overview of genetic studies on LD-associated proteins

Affected Species Diagnosis/genetic model protein Highlights of the study Reference

Human Partial lipodystrophy CIDEC Identiﬁcation of CIDEC truncation as Rubio-Cabezas et al. (2009) the causative mutation Human Partial lipodystrophy PLIN1 Identiﬁcation of two PLIN1 truncations Gandotra et al. (2011a) as causative mutations Human Partial lipodystrophy PLIN1 Characterization of PLIN1 mutants in Gandotra et al. (2011b) regulation of lipolysis in cell culture Mouse KO PLIN1 Phenotyping of KO animals Martinez-Botas et al. (2000) Mouse KO PLIN1 Phenotyping of KO animals Tansey et al. (2001) Mouse KO CIDEA Phenotyping of KO animals Zhou et al. (2003) Mouse KO CIDEB Phenotyping of KO animals Li et al. (2007) Mouse KO CIDEC Phenotyping of KO animals Nishino et al. (2008) Mouse KO CIDEC Analysis of WAT ‘browning’ in KO Toh et al. (2008) animals Mouse KO CIDEA Characterization of the role of CIDEA Zhou et al. (2012) in hepatic metabolism

Intracellular lipolysis in non-ATs DG to TG hydrolase activity (Jocken et al. 2010). Finally, in obese type 2 diabetic patients, basal SM lipolysis is elevated In this review, we have focused on lipolysis in AT. However, and the anti-lipolytic action of insulin is blunted (Jocken the ability to store and re-hydrolyze TG is not unique for et al. 2013). Whether this impairment is a result of peripheral adipocytes, and most cell types are able to form LDs by insulin resistance or a cause of it is unclear although, and at taking up and esterifying FFA into TG (Greenberg et al.2011). present, the mechanistic connection between intramyocel- In fact, the importance of tight lipolytic control in other lular lipolysis and insulin resistance is a matter of intense tissues has become apparent with the characterization of the debate and research. phenotypes associated with deficient lipase activity, particu- In summary, since the discovery of ATGL in 2004 larly with respect to ATGL. Like the myocardial defects tremendous progress has been made in the character- Atgl observed in KO mice, muscle-specific CGI-58 KO mice ization of AT lipolysis. However, with the increasing also suffer from cardiomyopathy and cardiac steatosis caused number of newly identified enzymes and regulatory by a severe impairment of TG catabolism (Zierler et al.2013). Journal of Molecular Endocrinology proteins, the remarkable complexity of the hormonal Conversely, myocardial specific ATGL overexpression in and intracellular signaling network regulating the lipo- type 1 diabetic mice confers protection against diabetes- lytic pathway has also become clear. Considering the induced cardiomyopathy (Pulinilkunnil et al. 2013), sup- severe phenotypes associated with defective lipolysis in porting a key role of ATGL in the regulation of metabolism AT, pancreas, liver, heart, and SM, it is evident that the and lipid homeostasis in the heart. Similar results have been balance between lipid mobilization, utilization, and obtained in the liver, where ATGL deficiency causes hepatic storage is crucial in most tissues. Consequently, by steatosis and reduced b-oxidation (Ong et al. 2011, Wu et al. delineating the processes regulating lipid metabolism in 2011) while liver-specific overexpression of ATGL or HSL adipose and non-ATs alike, we can also advance our reduces obesity-induced steatosis and increases b-oxidation understanding of glucose metabolism and identify new (Reid et al. 2008, Ong et al. 2011). Surprisingly although, in pathways to target in the treatment of metabolic disease spite of these effects of altered lipase expression on hepatic like type 2 diabetes. TG content and substrate utilization, liver-specific KO or overexpression of ATGL only has minor effects on hepatic

insulin sensitivity and whole-body metabolic parameters Declaration of interest (Turpin et al.2011, Wu et al. 2011). In human SM, ATGL The authors declare that there is no conﬂict of interest that could be perceived as prejudicing the impartiality of the review. protein is increased signiﬁcantly by endurance training, indicating that mobilization of intramyocellular TG stores involves ATGL (Alsted et al. 2009). Obesity is also associated Funding with increased SM content of ATGL, whereas HSL is This work was supported by grants from: i) The FOOD Study Group/Ministry decreased, resulting in a substantial decrease in the ratio of of Food, Agriculture and Fisheries & Ministry of Family and Consumer

Affairs, Denmark, ii) The Lundbeck Foundation, Denmark, iii) The Novo Bartz R, Zehmer JK, Zhu M, Chen Y, Serrero G, Zhao Y & Liu P 2007 Nordisk Foundation, Denmark, iv) Augustinus Fonden, Denmark, and Dynamic activity of lipid droplets: protein phosphorylation and v) Aase og Ejnar Danielsens Fond, Denmark. GTP-mediated protein translocation. Journal of Proteome Research 6 3256–3265. (doi:10.1021/pr070158j) Belfort R, Mandarino L, Kashyap S, Wirfel K, Pratipanawatr T, Berria R, DeFronzo RA & Cusi K 2005 Dose–response effect of elevated Acknowledgements plasma free fatty acid on insulin signaling. Diabetes 54 1640–1648. 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Received in ﬁnal form 14 February 2014 Accepted 25 February 2014 Accepted Preprint published online 27 February 2014