Advertisement

Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores

Open AccessPublished:October 23, 2008DOI:https://doi.org/10.1194/jlr.R800031-JLR200
      Fatty acids (FAs) are essential components of all lipid classes and pivotal substrates for energy production in all vertebrates. Additionally, they act directly or indirectly as signaling molecules and, when bonded to amino acid side chains of peptides, anchor proteins in biological membranes. In vertebrates, FAs are predominantly stored in the form of triacylglycerol (TG) within lipid droplets of white adipose tissue. Lipid droplet-associated TGs are also found in most nonadipose tissues, including liver, cardiac muscle, and skeletal muscle. The mobilization of FAs from all fat depots depends on the activity of TG hydrolases. Currently, three enzymes are known to hydrolyze TG, the well-studied hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL), discovered more than 40 years ago, as well as the relatively recently identified adipose triglyceride lipase (ATGL). The phenotype of HSL- and ATGL-deficient mice, as well as the disease pattern of patients with defective ATGL activity (due to mutation in ATGL or in the enzyme's activator, CGI-58), suggest that the consecutive action of ATGL, HSL, and MGL is responsible for the complete hydrolysis of a TG molecule. The complex regulation of these enzymes by numerous, partially uncharacterized effectors creates the “lipolysome,” a complex metabolic network that contributes to the control of lipid and energy homeostasis. This review focuses on the structure, function, and regulation of lipolytic enzymes with a special emphasis on ATGL.
      Lipid homeostasis reflects a balance of processes, designed to generate fatty acids (FAs) and lipids, deliver them from their site of origin to target tissues, and catabolize them for metabolic purposes. Innumerable genes and signal components are responsible for an integrated communication network between many tissues and organs, including adipose tissue, liver, muscles, the digestive tract, pancreas, and the nervous system. This network ultimately accounts for the accurate regulation of lipid and energy homeostasis. Despite the central physiological importance of these processes for human health, many basic mechanisms regulating the synthesis, uptake, storage, and utilization of lipids remain insufficiently characterized.
      FAs are vital components of essentially all known organisms. They are important substrates for oxidation and the production of cellular energy. FAs are essential precursors for all lipid classes, including those forming biological membranes. Finally, they are important for protein function in acylated proteins and as ligands for nuclear receptor transcription factors. In contrast to these “beneficial” characteristics, unesterified FAs can become deleterious for cells when present even at relatively low concentrations. The chronic exposure of nonadipose cells and tissues to elevated concentrations of FAs triggers adverse effects subsumed under the term of “lipotoxicity” (
      • Schaffer J.E
      Lipotoxicity: when tissues overeat..
      ,
      • Unger R.H
      Lipotoxic diseases..
      ). Accordingly, when supplied with excessive nutrients, essentially all eukaryotes reesterify and deposit FAs as triacylglycerol (TG) droplets to provide an energy reserve for times of nutrient deprivation and to detoxify otherwise harmful compounds.
      Until recently, lipid droplets were viewed as an inert storage pool of TG. It is now known that essentially all cells in the body generate lipid droplets composed of neutral lipids (TG and cholesteryl esters), phospholipids, and unesterified cholesterol at varying, tissue-specific concentrations. Additionally, numerous proteins are associated with lipid droplets (
      • Brasaemle D.L
      Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis..
      ,
      • Brasaemle D.L
      • Dolios G.
      • Shapiro L.
      • Wang R.
      Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3–L1 adipocytes..
      ,
      • Londos C.
      • Brasaemle D.L.
      • Schultz C.J.
      • Segrest J.P.
      • Kimmel A.R.
      Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells..
      ). These include structural proteins, lipid-modifying enzymes, and proteins that regulate enzyme activities. To date, the physiological role of many of these factors remains elusive. However, from the limited knowledge that is available, it is apparent that lipid droplets represent remarkably flexible, dynamic organelles that are used for the production of membrane components, energy substrates, and signaling molecules, including lipotoxic compounds (
      • Beckman M.
      Cell biology. Great balls of fat..
      ,
      • Martin S.
      • Parton R.G
      Lipid droplets: a unified view of a dynamic organelle..
      ). Although lipid droplets are observed in many cell types, the majority of fat in mammals is found in adipocytes of white adipose tissue (WAT). The central contribution of WAT to the regulation of energy homeostasis is due to both the enormous lipid storage capacity as well as its function as an endocrine organ secreting numerous hormones and adipo-cytokines (
      • Ahima R.S
      • Lazar M.A.
      Adipokines and the peripheral and neural control of energy balance..
      ). Prevalent metabolic diseases such as obesity and type 2 diabetes emerge when TG synthesis and catabolism lose synchrony.
      The key process in fat catabolism and the provision of energy substrate during times of nutrient deprivation (fasting) or enhanced energy demand (e.g., exercise) is the hydrolytic cleavage of stored TG, the generation of FAs and glycerol, and their release from adipocytes. A complex, hormonally controlled regulatory network controls the initiation of this process, called lipolysis, and ultimately activates key intracellular lipases to hydrolyze TG. Currently, three enzymes are known to have an established function in the lipolytic breakdown of fat in adipose and nonadipose tissues: adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL).

      REGULATION OF LIPOLYSIS

      Numerous lipolytic and antilipolytic effectors control the catabolism of stored fat in various tissues (
      • Langin D.
      Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome..
      ,
      • Holm C.
      • Osterlund T.
      • Laurell H.
      • Contreras J.A.
      Molecular mechanisms regulating hormone-sensitive lipase and lipolysis..
      ). These include hormones, cytokines, and adipokines. In adipose tissue, the most potent stimulatory signals are catecholamines acting on β-adrenergic receptors (
      • Lafontan M.
      • Berlan M.
      Fat cell adrenergic receptors and the control of white and brown fat cell function..
      ). Mouse adipocytes express three subtypes of β-adrenergic receptors (β-ARs): β1-AR, β2-AR, and β3-AR. In human adipose tissue, only β1 and β2 receptors induce lipolysis. When catecholamines bind to these receptors, stimulatory Gs proteins activate adenylate cyclase, causing a rise in cAMP levels and elevated activity of cAMP-dependent protein kinase-A (PKA) (
      • Holm C.
      • Osterlund T.
      • Laurell H.
      • Contreras J.A.
      Molecular mechanisms regulating hormone-sensitive lipase and lipolysis..
      ,
      • Collins S.
      • Cao W.
      • Robidoux J.
      Learning new tricks from old dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue metabolism..
      ,
      • Holm C.
      Molecular mechanisms regulating hormone-sensitive lipase and lipolysis..
      ). PKA-mediated phosphorlylation of target proteins, including lipolytic enzymes and lipid droplet-associated proteins, induces an increased release of FAs and glycerol from adipose tissue up to 100-fold. Other hormones that stimulate PKA via Gs protein-coupled receptors include glucagon, parathyroid hormone, thyrotropin, α-melanocyte-stimulating hormone, and adrenocorticotropin. Several antilipolytic factors have been shown to act through inhibitory Gi protein-coupled receptors (
      • Holm C.
      • Osterlund T.
      • Laurell H.
      • Contreras J.A.
      Molecular mechanisms regulating hormone-sensitive lipase and lipolysis..
      ). These factors include catecholamines acting through α2-adrenergic receptors (
      • Lafontan M.
      • Berlan M.
      Fat cell adrenergic receptors and the control of white and brown fat cell function..
      ), adenosine (A1-adenosine receptor) (
      • Larrouy D.
      • Galitzky J.
      • Lafontan M.
      A1 adenosine receptors in the human fat cell: tissue distribution and regulation of radioligand binding..
      ), prostaglandin (E2 receptor) (
      • Richelsen B.
      Release and effects of prostaglandins in adipose tissue..
      ), NPY (NPY-1 receptor) (
      • Bradley R.L
      • Mansfield J.P.
      • Maratos-Flier E.
      Neuropeptides, including neuropeptide Y and melanocortins, mediate lipolysis in murine adipocytes..
      ), and nicotinic acid (GPR109A receptor) (
      • Offermanns S.
      The nicotinic acid receptor GPR109A (HM74A or PUMA-G) as a new therapeutic target..
      ). The relative distribution of α- and β-adrenergic receptors therefore determines the lipolytic activity in a tissue- and cell type-specific manner.
      Insulin and insulin-like growth factor represent the most potent inhibitory hormones in lipolysis (
      • Langin D.
      Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome..
      ,
      • Degerman E.
      • Landstrom T.R.
      • Wijkander J.
      • Holst L.S.
      • Ahmad F.
      • Belfrage P.
      • Manganiello V.
      Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B..
      ). Their effects are primarily communicated through the insulin receptor (IR), polyphosphorylation of insulin receptor substrates 1–4 (IRS1–4), activation of phosphatidylinositol-3 kinase (PI3K), and the induction of the protein kinase B/AKT(PKB/AKT). Complexity in this essentially linear pathway is added by the divergence at so-called critical nodes that interact with other signaling cascades (
      • Taniguchi C.M
      • Emanuelli B.
      • Kahn C.R.
      Critical nodes in signalling pathways: insights into insulin action..
      ). Critical nodes in the IR pathway include the IR and IRS interacting with cytokine and extracellular signal-regulated kinase (ERK) signaling and PI3K activating both 3-phosphoinositide-dependent protein kinases (PDK1 and 2) as well as atypical protein kinases C (PKCλ and ζ). At this point, a signaling network is established that regulates innumerable biological processes (possibly more than 1,000). Lipolysis is affected in multiple steps, including the phosphorylation of phosphodiesterase 3B, causing the degradation of cAMP and loss of PKA activation (
      • Degerman E.
      • Landstrom T.R.
      • Wijkander J.
      • Holst L.S.
      • Ahmad F.
      • Belfrage P.
      • Manganiello V.
      Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B..
      ).
      The mechanisms through which other effectors regulate lipolysis are less well characterized. These include tumor necrosis factor-α (TNFα), growth hormone, the Cide domain-containing proteins (CideN) family of proteins (CIDEA, -B, and -C), and the CopI-ARF vesicle transport machinery described below.

      HSL, THE “CLASSIC” ENZYME IN LIPOLYSIS

      The first enzyme discovered to facilitate the hormone-induced catabolism of fat was HSL. Although the initial observations of fasting-induced lipolytic activity in WAT of dogs (
      • Quagliarello G.
      • Scoz G.
      The existence of a lipase in adipose tissue..
      ) and man (
      • Renold A.E
      • Marble A.
      Lipolytic activity of adipose tissue in man and rat..
      ) were reported as early as 1932 and 1950, respectively, it was not until the early 1960s that a WAT-associated lipase was shown to be regulated by hormones and found to be different from lipoprotein lipase (
      • Bjorntorp P.
      • Furman R.H.
      Lipolytic activity in rat heart..
      ,
      • Bjorntorp P.
      • Furman R.H.
      Lipolytic activity in rat epididymal fat pads..
      ,
      • Hollenberg C.H
      • Raben M.S.
      • Astwood E.B.
      The lipolytic response to corticotropin..
      ,
      • Rizack M.A
      Activation of an epinephrine-sensitive lipolytic activity from adipose tissue by adenosine 3′,5′-phosphate..
      ). In a landmark study, Vaughan, Berger, and Steinberg (
      • Vaughan M.
      • Berger J.E.
      • Steinberg D.
      Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue..
      ) discovered two independent lipolytic activities in WAT of various mammals and designated these enzymes HSL and MGL. The purification of HSL, cloning of the corresponding cDNA and gene, and high-level heterologous expression of the protein permitted an extensive study of the biochemical properties of the enzyme, its tissue-specific function, and its regulation by various agonists and antagonists. Several comprehensive reviews have been published recently to summarize these results (
      • Holm C.
      Molecular mechanisms regulating hormone-sensitive lipase and lipolysis..
      ,
      • Donsmark M.
      • Langfort J.
      • Holm C.
      • Ploug T.
      • Galbo H.
      Hormone-sensitive lipase as mediator of lipolysis in contracting skeletal muscle..
      ,
      • Haemmerle G.
      • Zimmermann R.
      • Zechner R.
      Letting lipids go: hormone-sensitive lipase..
      ,
      • Kraemer F.B
      • Shen W.J.
      Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis..
      ,
      • Yeaman S.J
      Hormone-sensitive lipase—new roles for an old enzyme..
      ).

      HSL enzymology

      HSL exhibits broad substrate specificity capable of hydolyzing TG, diacylglycerol (DG), monoacylglycerol (MG), cholesteryl esters (CEs), retinyl esters (REs), and other ester substrates such as p-nitrophenyl butyrate (
      • Yeaman S.J
      Hormone-sensitive lipase—a multipurpose enzyme in lipid metabolism..
      ). The relative maximal hydrolysis rates are in the range of 1: 10: 1: 4: 2 for TG: DG: MG:CE: RE. Thus, TGs are actually the worst substrate for HSL among all these natural lipid esters, whereas DGs are the best. HSL slightly favors unsaturated medium-chain FAs over saturated long-chain FAs in TG substrates (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ). However, the substrate specificity toward the length or saturation grade of acyl chains within lipid esters is not very pronounced. Within the TG molecule, HSL preferentially hydrolyzes primary ester bonds in the sn-1 and sn-3 positions (
      • Fredrikson G.
      • Belfrage P.
      Positional specificity of hormone-sensitive lipase from rat adipose tissue..
      ). Phosphorylation of HSL in vitro modestly increases enzyme activity for TG and CE hydrolysis by 1.5- to 2-fold (
      • Cook K.G
      • Yeaman S.J.
      • Stralfors P.
      • Fredrikson G.
      • Belfrage P.
      Direct evidence that cholesterol ester hydrolase from adrenal cortex is the same enzyme as hormone-sensitive lipase from adipose tissue..
      ,
      • Fredrikson G.
      • Stralfors P.
      • Nilsson N.O.
      • Belfrage P.
      Hormone-sensitive lipase of rat adipose tissue. Purification and some properties..
      ). The activity for DG or MG hydrolysis is not affected.

      HSL gene, mRNA, and protein structure

      The gene for human HSL (LIPE) spans a genomic region of 26 kb and is located on chromosome 19q13.2 (
      • Holm C.
      • Kirchgessner T.G.
      • Svenson K.L.
      • Fredrikson G.
      • Nilsson S.
      • Miller C.G.
      • Shively J.E.
      • Heinzmann C.
      • Sparkes R.S.
      • Mohandas T.
      • et al.
      Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3..
      ). In addition to 10 exons that are transcribed into HSL mRNA in all human and mouse tissues, alternative exon usage results in a significant variation in the 5′-region of HSL transcripts (
      • Blaise R.
      • Grober J.
      • Rouet P.
      • Tavernier G.
      • Daegelen D.
      • Langin D.
      Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins..
      ,
      • Blaise R.
      • Guillaudeux T.
      • Tavernier G.
      • Daegelen D.
      • Evrard B.
      • Mairal A.
      • Holm C.
      • Jegou B.
      • Langin D.
      Testis hormone-sensitive lipase expression in spermatids is governed by a short promoter in transgenic mice..
      ,
      • Grober J.
      • Laurell H.
      • Blaise R.
      • Fabry B.
      • Schaak S.
      • Holm C.
      • Langin D.
      Characterization of the promoter of human adipocyte hormone-sensitive lipase..
      ,
      • Langin D.
      • Laurell H.
      • Holst L.S.
      • Belfrage P.
      • Holm C.
      Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium..
      ). In adipose tissue, adrenal gland, and ovary, HSL transcription starts from multiple exons (exons A, B, C, D, or exon 1) within a 13 kb region. Because exons B, C, and D are noncoding, the alternative exon usage does not change the amino acid composition of the enzyme. In contrast, exon A contains coding information for 43 additional amino acids, leading to an alternative enzyme isoform. In testis, two tissue-specific exons (T1 and T2) are used as transcriptional start sites. Exon T1 codes for an additional 300 amino acids, whereas T2 contains no coding sequences. The high variability in exon usage results in various HSL mRNA and protein sizes in adipose tissue, pancreatic β-cells, ovaries, and testis. Multiple potential transcription factor binding elements upstream of each transcriptional start site suggest the possibility of differential transcriptional regulation of HSL in different tissues and under various physiological conditions.
      According to the HSL domain structure model [the three-dimensional (3D) structure of the enzyme remains to be elucidated], the enzyme can be subdivided into three functional regions (
      • Holm C.
      • Davis R.C.
      • Osterlund T.
      • Schotz M.C.
      • Fredrikson G.
      Identification of the active site serine of hormone-sensitive lipase by site-directed mutagenesis..
      ,
      • Osterlund T.
      • Beussman D.J.
      • Julenius K.
      • Poon P.H.
      • Linse S.
      • Shabanowitz J.
      • Hunt D.F.
      • Schotz M.C.
      • Derewenda Z.S.
      • Holm C.
      Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry..
      ,
      • Osterlund T.
      • Contreras J.A.
      • Holm C.
      Identification of essential aspartic acid and histidine residues of hormone-sensitive lipase: apparent residues of the catalytic triad..
      ,
      • Osterlund T.
      • Danielsson B.
      • Degerman E.
      • Contreras J.A.
      • Edgren G.
      • Davis R.C.
      • Schotz M.C.
      • Holm C.
      Domain-structure analysis of recombinant rat hormone-sensitive lipase..
      ). The N-terminal domain (amino acids 1–300) is believed to mediate enzyme dimerization (
      • Shen W.J
      • Patel S.
      • Hong R.
      • Kraemer F.B.
      Hormone-sensitive lipase functions as an oligomer..
      ) and interaction with FABP4, a fatty acid binding protein known to enhance HSL enzyme activity (
      • Shen W.J
      • Liang Y.
      • Hong R.
      • Patel S.
      • Natu V.
      • Sridhar K.
      • Jenkins A.
      • Bernlohr D.A.
      • Kraemer F.B.
      Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase..
      ,
      • Shen W.J
      • Sridhar K.
      • Bernlohr D.A.
      • Kraemer F.B.
      Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein..
      ,
      • Smith A.J
      • Thompson B.R.
      • Sanders M.A.
      • Bernlohr D.A.
      Interaction of the adipocyte fatty acid-binding protein with the hormone-sensitive lipase: regulation by fatty acids and phosphorylation..
      ). The C-terminal domain contains the catalytic triad composed of serine 423, aspartate 703, and histidine 733 (numbering relates to rat HSL, isoform 2) within an α/β hydrolase fold typically found in many lipases and esterases. The third domain represents the regulatory module of the enzyme. This loop region (amino acids 521–669) contains all known phosphorylation sites of HSL.

      HSL regulation of enzyme activity

      Two major mechanisms determine HSL activity: enzyme phosphorylation by protein kinases and interaction with auxiliary proteins. The pathway of β-adrenergic stimulation involves the PKA-mediated phosphorylation of HSL. Originally it was believed that phosphorylation at two serine residues (563 and 565) (numbering relates to rat HSL, isoform 2) was sufficient to mediate the cAMP-dependent activation of HSL (
      • Stralfors P.
      • Belfrage P.
      Phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase..
      ,
      • Stralfors P.
      • Bjorgell P.
      • Belfrage P.
      Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin..
      ). Serine 565 was considered the basal phosphorylation site and serine 563 the regulatory site (
      • Garton A.J
      • Campbell D.G.
      • Cohen P.
      • Yeaman S.J.
      Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase..
      ,
      • Garton A.J
      • Yeaman S.J.
      Identification and role of the basal phosphorylation site on hormone-sensitive lipase..
      ,
      • Olsson H.
      • Belfrage P.
      The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B..
      ). However, PKA-mediated enzyme activation in an HSL variant in which Ser 563 was replaced by alanine led to the discovery of additional PKA phosphorylation sites (
      • Anthonsen M.W
      • Ronnstrand L.
      • Wernstedt C.
      • Degerman E.
      • Holm C.
      Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro..
      ). The identification of these additional serines that are targets for phosphorylation by PKA (Ser 659 and Ser 660) (
      • Anthonsen M.W
      • Ronnstrand L.
      • Wernstedt C.
      • Degerman E.
      • Holm C.
      Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro..
      ), ERK (Ser 600) (
      • Greenberg A.S
      • Shen W.J.
      • Muliro K.
      • Patel S.
      • Souza S.C.
      • Roth R.A.
      • Kraemer F.B.
      Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway..
      ), glycogen synthase kinase-4 (Ser 563) (
      • Olsson H.
      • Stralfors P.
      • Belfrage P.
      Phosphorylation of the basal site of hormone-sensitive lipase by glycogen synthase kinase-4..
      ), Ca2+/calmodulin-dependent kinase II (Ser 565) (
      • Garton A.J
      • Campbell D.G.
      • Carling D.
      • Hardie D.G.
      • Colbran R.J.
      • Yeaman S.J.
      Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism..
      ), and AMP-activated kinase (Ser 565) (
      • Garton A.J
      • Campbell D.G.
      • Carling D.
      • Hardie D.G.
      • Colbran R.J.
      • Yeaman S.J.
      Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism..
      ) has markedly increased the complexity of posttranslational HSL modification and regulation. Enzymes involved in the dephosphorylation of HSL include protein phosphatases 1, 2A, and 2C (
      • Olsson H.
      • Belfrage P.
      Phosphorylation and dephosphorylation of hormone-sensitive lipase. Interactions between the regulatory and basal phosphorylation sites..
      ).
      HSL phosphorylation by PKA in response to β-adrenergic stimulation induces the intrinsic HSL enzyme activity only moderately (approximately 2-fold). This is in sharp contrast to findings in intact cells where β-adrenergic stimulation and activation of PKA cause up to a 100-fold induction of FA and glycerol release. Thus, in addition to HSL modification, other mechanisms must contribute to hormone-induced lipolysis. This finding led to the discovery of perilipin (
      • Brasaemle D.L
      Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis..
      ,
      • Granneman J.G
      • Moore H.P.
      Location, location: protein trafficking and lipolysis in adipocytes..
      ,
      • Londos C.
      • Sztalryd C.
      • Tansey J.T.
      • Kimmel A.R.
      Role of PAT proteins in lipid metabolism..
      ,
      • Tansey J.T
      • Sztalryd C.
      • Hlavin E.M.
      • Kimmel A.R.
      • Londos C.
      The central role of perilipin a in lipid metabolism and adipocyte lipolysis..
      ). Perilipin is expressed mostly in WAT and steroidogenic tissues, where it localizes to the surface of lipid droplets (
      • Greenberg A.S
      • Egan J.J.
      • Wek S.A.
      • Garty N.B.
      • Blanchette-Mackie E.J.
      • Londos C.
      Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets..
      ). β-adrenergic stimulation of adipocytes causes the PKA-mediated polyphosphorylation of six defined serine residues within the protein (Ser 81, -222, -276, -433, -492, and -517), which results in the translocation of HSL to the lipid droplet and initiation of hydrolysis (
      • Miyoshi H.
      • Perfield II, J.W
      • Souza S.C.
      • Shen W.J.
      • Zhang H.H.
      • Stancheva Z.S.
      • Kraemer F.B.
      • Obin M.S.
      • Greenberg A.S.
      Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes..
      ,
      • Miyoshi H.
      • Souza S.C.
      • Zhang H.H.
      • Strissel K.J.
      • Christoffolete M.A.
      • Kovsan J.
      • Rudich A.
      • Kraemer F.B.
      • Bianco A.C.
      • Obin M.S.
      • et al.
      Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms..
      ,
      • Sztalryd C.
      • Xu G.
      • Dorward H.
      • Tansey J.T.
      • Contreras J.A.
      • Kimmel A.R.
      • Londos C.
      Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation..
      ,
      • Tansey J.T
      • Huml A.M.
      • Vogt R.
      • Davis K.E.
      • Jones J.M.
      • Fraser K.A.
      • Brasaemle D.L.
      • Kimmel A.R.
      • Londos C.
      Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols..
      ). Although originally HSL binding to the lipid droplet was seen in association with perilipin dissociation, the characterization of perilipin-deficient mice and functional studies with perilipin mutants redefined and extended this “replacement” hypothesis (
      • Clifford G.M
      • Londos C.
      • Kraemer F.B.
      • Vernon R.G.
      • Yeaman S.J.
      Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes..
      ,
      • Martinez-Botas J.
      • Anderson J.B.
      • Tessier D.
      • Lapillonne A.
      • Chang B.H.
      • Quast M.J.
      • Gorenstein D.
      • Chen K.H.
      • Chan L.
      Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice..
      ,
      • Tansey J.T
      • Sztalryd C.
      • Gruia-Gray J.
      • Roush D.L.
      • Zee J.V.
      • Gavrilova O.
      • Reitman M.L.
      • Deng C.X.
      • Li C.
      • Kimmel A.R.
      • et al.
      Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity..
      ). During hormone stimulation, perilipin is essential for the recruitment of HSL to lipid droplets and full enzyme activation (
      • Sztalryd C.
      • Xu G.
      • Dorward H.
      • Tansey J.T.
      • Contreras J.A.
      • Kimmel A.R.
      • Londos C.
      Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation..
      ). Notably, perilipin phosphorylation is not required for the translocation of HSL, because unphosphorylated perilipin mutants still recruit HSL to the surface of lipid droplets (
      • Miyoshi H.
      • Souza S.C.
      • Zhang H.H.
      • Strissel K.J.
      • Christoffolete M.A.
      • Kovsan J.
      • Rudich A.
      • Kraemer F.B.
      • Bianco A.C.
      • Obin M.S.
      • et al.
      Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms..
      ). In contrast, perilipin phosphorylation is absolutely crucial for the hydrolytic activity of HSL. Perilipin is mostly associated with small lipid droplets within fat cells and, in fact, contributes to the fragmentation of large lipid droplets during the lipolytic process. The latter activity of perilipin involves its phosphorylation at serine residue 492 (
      • Marcinkiewicz A.
      • Gauthier D.
      • Garcia A.
      • Brasaemle D.L.
      The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion..
      ,
      • Moore H.P
      • Silver R.B.
      • Mottillo E.P.
      • Bernlohr D.A.
      • Granneman J.G.
      Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase..
      ).
      Perilipin belongs to the PAT family of proteins (as reviewed in Refs.
      • Brasaemle D.L
      Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis..
      ,
      • Ducharme N.A
      • Bickel P.E.
      Lipid droplets in lipogenesis and lipolysis..
      ). These factors include perilipin, adipophilin (ADRP), Tip47, S3-12, and myocyte lipid droplet protein (MLDP, also termed OXPAT). Because perilipin expression is mostly restricted to adipose and steroidogenic cells, it is of great interest to determine whether other PAT family members can accomplish a similar regulatory role for HSL in nonadipose tissues. In one report, HSL interaction with lipotransin was shown to activate HSL-mediated lipolysis (
      • Syu L.J
      • Saltiel A.R.
      Lipotransin: a novel docking protein for hormone-sensitive lipase..
      ). However, this mechanism has not been confirmed since its original observation.

      HSL deficiency in mice: HSL is not alone

      For more than three decades, HSL was considered to be the only and therefore rate-limiting enzyme for the lipolytic catabolism of stored fat in adipose and nonadipose tissues. Because HSL was shown to hydrolyze both TG and DG substrates, it was believed that the enzyme represented the only lipase activated by hormonal stimulation. This view, however, changed when several independent laboratories reported on the phenotype of HSL-deficient mice (
      • Haemmerle G.
      • Zimmermann R.
      • Strauss J.G.
      • Kratky D.
      • Riederer M.
      • Knipping G.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle..
      ,
      • Osuga J.
      • Ishibashi S.
      • Oka T.
      • Yagyu H.
      • Tozawa R.
      • Fujimoto A.
      • Shionoiri F.
      • Yahagi N.
      • Kraemer F.B.
      • Tsutsumi O.
      • et al.
      Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity..
      ,
      • Wang S.P
      • Laurin N.
      • Himms-Hagen J.
      • Rudnicki M.A.
      • Levy E.
      • Robert M.F.
      • Pan L.
      • Oligny L.
      • Mitchell G.A.
      The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice..
      ). Although HSL deficiency causes infertility in male mice, owing to a defect in sperm maturation, the animals are normal with regard to their lipid and energy metabolism. Unexpectedly, HSL knockout (HSL-ko) mice were not overweight or obese. To the contrary, with increased age, they had reduced WAT weight (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ) and were resistant to genetically or diet-induced obesity (
      • Harada K.
      • Shen W.J.
      • Patel S.
      • Natu V.
      • Wang J.
      • Osuga J.
      • Ishibashi S.
      • Kraemer F.B.
      Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice..
      ). HSL-deficient adipocytes responded to β-adrenergic stimulation and, compared with control mice, exhibited only a moderate decrease in their capacity to release FA (∼40%) (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ,
      • Okazaki H.
      • Osuga J.
      • Tamura Y.
      • Yahagi N.
      • Tomita S.
      • Shionoiri F.
      • Iizuka Y.
      • Ohashi K.
      • Harada K.
      • Kimura S.
      • et al.
      Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases..
      ). Importantly, HSL deficiency resulted in DG accumulation in several tissues, indicating that HSL is rate-limiting for DG hydrolysis (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ). These findings strongly suggested that at least one additional enzyme acted as TG hydrolase when HSL was absent and that this activity was either directly or indirectly “hormone sensitive.” The findings also indicated that HSL was more important as DG hydrolase than as TG hydrolase.

      ATGL: A NEW PLAYER IN THE LIPOLYSIS TEAM

      In 2004, three groups independently published the discovery of an enzyme able to hydrolyze TG and named it ATGL (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ), desnutrin (
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ), or calcium-independent phospholipase A2ζ (iPLA2ζ) (
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ). Very soon after these initial reports, ATGL orthologous genes and proteins were identified and characterized in other vertebrates, flies, fungi, and plants (
      • Eastmond P.J
      SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds..
      ,
      • Gronke S.
      • Mildner A.
      • Fellert S.
      • Tennagels N.
      • Petry S.
      • Muller G.
      • Jackle H.
      • Kuhnlein R.P.
      Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila..
      ,
      • Kurat C.F
      • Natter K.
      • Petschnigg J.
      • Wolinski H.
      • Scheuringer K.
      • Scholz H.
      • Zimmermann R.
      • Leber R.
      • Zechner R.
      • Kohlwein S.D.
      Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast..
      ,
      • Saarela J.
      • Jung G.
      • Hermann M.
      • Nimpf J.
      • Schneider W.J.
      The patatin-like lipase family in Gallus gallus..
      ,
      • Shan T.
      • Wang Y.
      • Wu T.
      • Guo J.
      • Liu J.
      • Feng J.
      • Xu Z.
      Porcine adipose triglyceride lipase complementary deoxyribonucleic acid clone, expression pattern, and regulation by resveratrol..
      ). Work with the Drosophila melanogaster enzyme “brummer,” triacylglycerol lipase-4 in Saccharomyces cerevisiae, and sugar-dependent1 in Arabidopsis thaliana demonstrated that each of these proteins exhibits robust TG hydrolase activity and has a fundamental role in the regulation of TG homeostasis in the respective organism.

      ATGL enzymology

      ATGL exhibits 10-fold higher substrate specificity for TG than for DG and selectively performs the first step in TG hydrolysis, resulting in the formation of DG and FA (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ). The stereospecificity of ATGL for the chemically distinct ester bonds within the TG molecule is currently not known. Therefore, it is also unclear whether the DG generated by ATGL can participate in signaling processes involving sn-1,2-DG, such as the activation of various PKC isoenzymes. ATGL was also reported to have transacylase (
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ) and phospholipase activity (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Notari L.
      • Baladron V.
      • Aroca-Aguilar J.D.
      • Balko N.
      • Heredia R.
      • Meyer C.
      • Notario P.M.
      • Saravanamuthu S.
      • Nueda M.L.
      • Sanchez-Sanchez F.
      • et al.
      Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor..
      ) that was shown to be lower than its TG hydrolase activity (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ). In contrast to HSL, ATGL does not hydrolyze MG, CE, or RE. Smirnova et al. (
      • Smirnova E.
      • Goldberg E.B.
      • Makarova K.S.
      • Lin L.
      • Brown W.J.
      • Jackson C.L.
      ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells..
      ) demonstrated that the hydrolytic function of ATGL is not restricted to the catabolism of lipid droplets (“adiposomes”) in adipose tissue and suggested the enzyme be renamed adiposome triglyceride lipase rather than adipose triglyceride lipase to more adequately reflect its function.

      ATGL gene, mRNA, and protein structure

      The mouse Atgl gene (Pnpla2) contains nine exons and spans a region of approximately 6 kb on chromosome 7F5. Transcription of the gene results in a 1.96 kb mRNA coding for a 486 amino acid protein with a molecular mass of 54 kDa. The ten exons of the human ATGL gene (PNPLA2) span 6.32 kb of genomic DNA, which are located on chromosome 11p15.5. Mammalian ATGL belongs to a gene family characterized by the presence of a patatin domain (Pfam01734). This structural motif was designated for patatin, the most abundant protein in the potato tuber, with established DG, MG, and phospholipase activity, but no TG hydrolase activity (
      • Andrews D.L
      • Beames B.
      • Summers M.D.
      • Park W.D.
      Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector..
      ,
      • Jimenez-Atienzar M.
      • Cabanes J.
      • Gandia-Herrero F.
      • Escribano J.
      • Garcia-Carmona F.
      • Perez-Gilabert M.
      Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay..
      ,
      • Senda K.
      • Yoshioka H.
      • Doke N.
      • Kawakita K.
      A cytosolic phospholipase A2 from potato tissues appears to be patatin..
      ). ATGL is most closely related to a group of five genes and proteins named patatin-like phospholipase domain-containing 1 to 5 (PNPLA1-5) (
      • Wilson P.A
      • Gardner S.D.
      • Lambie N.M.
      • Commans S.A.
      • Crowther D.J.
      Characterization of the human patatin-like phospholipase family..
      ,
      • Zechner R.
      • Strauss J.G.
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      Lipolysis: pathway under construction..
      ). Members of this protein family in addition to ATGL (PNPLA2) are PNPLA1, adiponutrin (PNPLA3), GS2 (PNPLA4), and GS2-like (PNPLA5). To date, no orthologous gene for GS2 has been identified in the mouse genome. More distantly related members of ATGL include neuropathy target esterase (NTE, PNPLA6), NTE-related esterase (NRE, PNPLA7), calcium-independent phospholipase A2γ (iPLA2γ, PNPLA8), and phospholipase A2 group VI (PLA2G6, PNPLA9). Like ATGL, adiponutrin, GS2, and GS2-like also exhibit hydrolase and transacylase activity in in vitro assays (
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ). Low specific phospholipase activity was reported for ATGL, adiponutrin, and GS2-like (
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Notari L.
      • Baladron V.
      • Aroca-Aguilar J.D.
      • Balko N.
      • Heredia R.
      • Meyer C.
      • Notario P.M.
      • Saravanamuthu S.
      • Nueda M.L.
      • Sanchez-Sanchez F.
      • et al.
      Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor..
      ). Considering the structural and functional diversity within patatin domain-containing proteins, the name patatin-like phospholipase domain-containing 1-9 for these proteins is somewhat misleading and should be changed to a more general name such as patatin domain-containing lipid hydrolase 1-9(PDLH1-9).
      The primary structures of the human and murine ATGL enzyme share 84% sequence identity. Sequence identity is particularly high within the patatin domain (>95%) harboring the active site of the enzyme. A schematic representation of the domain structure of ATGL is shown in Fig. 1. Interestingly, unlike other typical TG hydrolases, the active site of patatin domain-containing enzymes is not composed of a catalytic triad. Instead, 3D structure determination of related members of the family (potato patatin) revealed that the enzyme mechanism depends on a catalytic dyad (
      • Rydel T.J
      • Williams J.M.
      • Krieger E.
      • Moshiri F.
      • Stallings W.C.
      • Brown S.M.
      • Pershing J.C.
      • Purcell J.P.
      • Alibhai M.F.
      The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad..
      ). In ATGL, mutational analyses identified serine 47 as the active site nucleophile located within a canonical GXSXG sequence (
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ,
      • Smirnova E.
      • Goldberg E.B.
      • Makarova K.S.
      • Lin L.
      • Brown W.J.
      • Jackson C.L.
      ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells..
      ,
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ). From homology considerations, it is assumed that aspartate 166 is the second amino acid critical for the catalytic dyad in ATGL. Sequence and 3D-structural similarities also indicate that the dyad is embedded within a three-layer α/β/α architecture commonly found in hydrolases/esterases (
      • Schneider G.
      • Neuberger G.
      • Wildpaner M.
      • Tian S.
      • Berezovsky I.
      • Eisenhaber F.
      Application of a sensitive collection heuristic for very large protein families: evolutionary relationship between adipose triglyceride lipase (ATGL) and classic mammalian lipases..
      ). Similarly, as has been shown for other patatin domain enzymes, the transition state in ATGL might be stabilized by a glycine-rich oxyanion hole. The C-terminal region of ATGL exhibits only poor homology to the other members of the PNPLA family. A hydrophobic stretch from amino acids 315 to 360 was proposed to mediate lipid droplet binding (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ). Additionally, two phosphorylation sites were identified in the C-terminal region of the enzyme (serine 404 and serine 428 in human ATGL) (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Bartz R.
      • Zehmer J.K.
      • Zhu M.
      • Chen Y.
      • Serrero G.
      • Zhao Y.
      • Liu P.
      Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation..
      ). The functional roles of enzyme phosphorylation and involved protein kinases remain unknown. Notably, the human protein is 19 amino acids longer than the mouse ortholog and contains a proline-rich sequence on its very C terminus. Whether this peptide stretch contributes to species-specific differences in ATGL regulation and function requires clarification.
      Figure thumbnail gr1
      Fig. 1Representation of the structural domains of human adipose triglyceride lipase (ATGL) protein. The crucial structural components for enzyme function are indicated, including the patatin domain, α/β hydrolase region, active site serine (S47), putative aspartic acid within the catalytic dyad (D166), potential lipid binding domain (hydrophobic), and two established phosphorylation sites (serine 404 and serine 428). Mutations associated with neutral lipid storage disease with myopathy are also indicated.

      ATGL physiological function

      The important role of ATGL in lipolysis became evident from observations in ATGL-deficient (ATGL-ko) mice (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ). In contrast to HSL-deficient mice, ATGL-ko animals had a severe “lipid” phenotype (for phenotype comparison of HSL-ko and ATGL-ko mice, see Table 1). Absence of ATGL causes a reduction of FA release from WAT by more than 75%. ATGL-ko mice accumulated TG in essentially all organs and cell types analyzed, consistent with an important function of ATGL in TG catabolism in multiple tissues. Defective TG mobilization and massive TG accumulation cause severe myopathy in cardiac muscle, defective thermogenesis in brown adipose tissue (BAT), and an overall defect in energy homeostasis (see section on the tissue-specific function of ATGL below). The excessive TG accumulation in the heart causes cardiac dysfunction and premature death in ATGL-deficient mice. In WAT, TG hydrolase activity and hormone-stimulated lipolysis were also drastically reduced, which is consistent with the view that ATGL is “hormone sensitive” via either a direct or an indirect mechanism.
      TABLE 1Phenotypes observed in ATGL-deficient and HSL-deficient mice
      ParameterATGL-koHSL-ko
      Life spanReducedNormal
      FertilityNormalInfertile
      Body weightIncreasedNormal
      Fat massIncreasedReduced
      Fat accumulation in nonadipose tissuesSevereReduced
      Tissue DG accumulationNormalSevere
      ThermogenesisDefectiveNormal
      Plasma FAReducedReduced
      Plasma TGReducedReduced
      Plasma ketone bodiesReducedReduced
      Plasma cholesterolReducedIncreased
      Plasma HDL cholesterolReducedIncreased
      Glucose/insulin toleranceIncreasedIncreased
      ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; ko, knockout; DG, diacylglycerol; TG, triacylglycerol. Data assembled from (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ,
      • Haemmerle G.
      • Zimmermann R.
      • Strauss J.G.
      • Kratky D.
      • Riederer M.
      • Knipping G.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle..
      ,
      • Osuga J.
      • Ishibashi S.
      • Oka T.
      • Yagyu H.
      • Tozawa R.
      • Fujimoto A.
      • Shionoiri F.
      • Yahagi N.
      • Kraemer F.B.
      • Tsutsumi O.
      • et al.
      Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity..
      ,
      • Wang S.P
      • Laurin N.
      • Himms-Hagen J.
      • Rudnicki M.A.
      • Levy E.
      • Robert M.F.
      • Pan L.
      • Oligny L.
      • Mitchell G.A.
      The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice..
      ,
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ).
      In contrast to HSL-ko animals, ATGL-deficient male mice are fertile, indicating that massive TG accumulation in testis per se is not causative for male infertility. Instead, it appears more likely that the impaired hydrolysis of DG, CE, and RE may cause a defect in germ cell maturation in HSL-deficient mice. The concentration of plasma FA, TG, and ketone bodies are decreased in both fasted HSL-ko and ATGL-ko mice, yet the absolute levels are lower in ATGL deficiency and are also decreased in fed ATGL-ko animals as compared with wild-type littermates. Interestingly, total cholesterol and HDL cholesterol concentrations are elevated in HSL-deficient mice and reduced in ATGL-deficient mice. The reason for this unexpected difference is unclear and requires elucidation.
      Taken together, the analysis of ATGL-ko mice suggested that ATGL is rate-limiting for the first step in TG hydrolysis, generating DG and FA with an approximately 10-fold higher specificity toward TG than HSL (calculated). HSL efficiently degrades DG, generating MG and FA. The final step, resulting in the formation of glycerol and FA, is performed by MGL. Whether other TG hydrolases in addition to ATGL and HSL also contribute to the hydrolysis of TG in WAT was recently addressed by Schweiger et al. (
      • Schweiger M.
      • Schreiber R.
      • Haemmerle G.
      • Lass A.
      • Fledelius C.
      • Jacobsen P.
      • Tornqvist H.
      • Zechner R.
      • Zimmermann R.
      Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism..
      ). Complete inhibition of HSL with a specific inhibitor (provided by Novo Nordisk) resulted in an almost complete absence of FA release in ATGL-deficient adipose tissue, suggesting that besides ATGL and HSL, additional lipases contribute little to the lipolytic capacity of white fat cells in mice. The role of alternative lipases, such as Ces3 (
      • Soni K.G
      • Lehner R.
      • Metalnikov P.
      • O'Donnell P.
      • Semache M.
      • Gao W.
      • Ashman K.
      • Pshezhetsky A.V.
      • Mitchell G.A.
      Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase..
      ) or TGH-2 (
      • Okazaki H.
      • Igarashi M.
      • Nishi M.
      • Tajima M.
      • Sekiya M.
      • Okazaki S.
      • Yahagi N.
      • Ohashi K.
      • Tsukamoto K.
      • Amemiya-Kudo M.
      • et al.
      Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis..
      ), in WAT under specific physiological conditions or their contribution to lipolysis in nonadipose tissues remains to be determined.

      Regulation of ATGL: hormones and cytokines

      Although ATGL is expressed in most tissues of the body, the highest levels of mRNA and enzyme activity are found in WAT and BAT (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ,
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ,
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Jocken J.W
      • Langin D.
      • Smit E.
      • Saris W.H.
      • Valle C.
      • Hul G.B.
      • Holm C.
      • Arner P.
      • Blaak E.E.
      Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state..
      ,
      • Langin D.
      • Dicker A.
      • Tavernier G.
      • Hoffstedt J.
      • Mairal A.
      • Ryden M.
      • Arner E.
      • Sicard A.
      • Jenkins C.M.
      • Viguerie N.
      • et al.
      Adipocyte lipases and defect of lipolysis in human obesity..
      ,
      • Mairal A.
      • Langin D.
      • Arner P.
      • Hoffstedt J.
      Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity..
      ,
      • Ryden M.
      • Jocken J.
      • van Harmelen V.
      • Dicker A.
      • Hoffstedt J.
      • Wiren M.
      • Blomqvist L.
      • Mairal A.
      • Langin D.
      • Blaak E.
      • et al.
      Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis..
      ,
      • Steinberg G.R
      • Kemp B.E.
      • Watt M.J.
      Adipocyte triglyceride lipase expression in human obesity..
      ). During adipocyte differentiation of 3T3-L1 cells, ATGL expression is strongly induced, reaching maximal levels when the cells accumulate visible lipid droplets (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ,
      • Jenkins C.M
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities..
      ,
      • Notari L.
      • Baladron V.
      • Aroca-Aguilar J.D.
      • Balko N.
      • Heredia R.
      • Meyer C.
      • Notario P.M.
      • Saravanamuthu S.
      • Nueda M.L.
      • Sanchez-Sanchez F.
      • et al.
      Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor..
      ,
      • Wilson P.A
      • Gardner S.D.
      • Lambie N.M.
      • Commans S.A.
      • Crowther D.J.
      Characterization of the human patatin-like phospholipase family..
      ,
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Kershaw E.E
      • Hamm J.K.
      • Verhagen L.A.
      • Peroni O.
      • Katic M.
      • Flier J.S.
      Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin..
      ). Compared with WAT and BAT, ATGL mRNA levels are much lower in other tissues. Quantitative PCR analysis revealed that adrenals, testis, cardiac muscle, and skeletal muscle have approximately 25% of the ATGL mRNA levels (normalized to tissue protein content) found in WAT, other tissues around 10% (
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ,
      • Kershaw E.E
      • Hamm J.K.
      • Verhagen L.A.
      • Peroni O.
      • Katic M.
      • Flier J.S.
      Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin..
      ).
      In contrast to a wealth of available information on the regulation of HSL in WAT, comparatively little is known about the molecular pathways leading to the activation of ATGL activity. The ability of HSL-ko WAT to respond to hormonal stimulation (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ) and the finding that HSL inhibition in WAT leaves a “hormone-inducible” hydrolytic activity (
      • Schweiger M.
      • Schreiber R.
      • Haemmerle G.
      • Lass A.
      • Fledelius C.
      • Jacobsen P.
      • Tornqvist H.
      • Zechner R.
      • Zimmermann R.
      Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism..
      ) suggest that ATGL activity is either directly or indirectly activated by hormonal signals. Several observations indicate that the molecular mechanism leading to ATGL activation is different from that described for HSL. First, unlike HSL, ATGL is present on lipid droplets of adipocytes in similar amounts in the basal state and in the activated state (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ). Second, although ATGL can be phosphorylated, it is not a target for PKA (
      • Zimmermann R.
      • Strauss J.G.
      • Haemmerle G.
      • Schoiswohl G.
      • Birner-Gruenberger R.
      • Riederer M.
      • Lass A.
      • Neuberger G.
      • Eisenhaber F.
      • Hermetter A.
      • et al.
      Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase..
      ,
      • Bartz R.
      • Zehmer J.K.
      • Zhu M.
      • Chen Y.
      • Serrero G.
      • Zhao Y.
      • Liu P.
      Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation..
      ). Third, ATGL activity is greatly enhanced by a protein annotated as α/β hydrolase domain-containing protein 5 (ABHD5) or comparative gene identification-58 (CGI-58) (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ). CGI-58 does not affect HSL enzyme activity (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ).
      To date, most studies addressing the regulation of ATGL by hormonal or nutritional effectors have restricted their analyses to the measurement of ATGL mRNA levels and have not reported ATGL enzyme activities. Considering the likely posttranscriptional regulation of ATGL and HSL by phosphorylation and numerous modulating protein factors (see below), this is unfortunate, and conclusions drawn from these data must be viewed with caution when lipolytic activities are assumed from lipase mRNA concentrations. As expected for a TG hydrolase active in WAT, ATGL mRNA concentrations are markedly affected by nutritional status, and increase during fasting and decrease during refeeding (
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ,
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ,
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Kershaw E.E
      • Hamm J.K.
      • Verhagen L.A.
      • Peroni O.
      • Katic M.
      • Flier J.S.
      Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin..
      ). ATGL mRNA levels during fasting are not paralleled by HSL mRNA levels that are downregulated during acute fasting and increase only after prolonged food deprivation (3–5 days) (
      • Sztalryd C.
      • Kraemer F.B.
      Regulation of hormone-sensitive lipase during fasting..
      ). From in vitro experiments in murine 3T3-L1 adipocytes, Villena et al. (
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ) concluded that glucocorticoids could be responsible for the increase of ATGL mRNA levels in the fasted state. The observation that ATGL mRNA is significantly downregulated in genetic models of obesity (ob/ob and db/db mice) suggested a possible contribution of ATGL in the pathogenesis of obesity (
      • Villena J.A
      • Roy S.
      • Sarkadi-Nagy E.
      • Kim K.H.
      • Sul H.S.
      Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis..
      ,
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ); however, this effect was not observed in all studies (
      • Lake A.C
      • Sun Y.
      • Li J.L.
      • Kim J.E.
      • Johnson J.W.
      • Li D.
      • Revett T.
      • Shih H.H.
      • Liu W.
      • Paulsen J.E.
      • et al.
      Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members..
      ).
      The enormous induction of FA and glycerol release from fat cells in response to β-adrenergic stimulation is not associated with increased levels of either ATGL or HSL mRNA. In fact, in some studies, isoproterenol treatment of 3T3-L1 cells or isolated adipocytes causes decreased mRNA concentrations of both lipases (
      • Kralisch S.
      • Klein J.
      • Lossner U.
      • Bluher M.
      • Paschke R.
      • Stumvoll M.
      • Fasshauer M.
      Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes..
      ,
      • Slavin B.G
      • Ong J.M.
      • Kern P.A.
      Hormonal regulation of hormone-sensitive lipase activity and mRNA levels in isolated rat adipocytes..
      ,
      • Plee-Gautier E.
      • Grober J.
      • Duplus E.
      • Langin D.
      • Forest C.
      Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes..
      ). This suggests that the β-adrenergic stimulation of lipolysis is exclusively regulated posttranscriptionally.
      Insulin treatment reduces ATGL mRNA levels in murine 3T3-L1 adipocytes (
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Kershaw E.E
      • Hamm J.K.
      • Verhagen L.A.
      • Peroni O.
      • Katic M.
      • Flier J.S.
      Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin..
      ,
      • Kralisch S.
      • Klein J.
      • Lossner U.
      • Bluher M.
      • Paschke R.
      • Stumvoll M.
      • Fasshauer M.
      Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes..
      ). Importantly, this inhibitory effect of insulin on ATGL expression was also demonstrated in vivo using mouse models of systemic insulin deficiency (streptozotocin-treated animals) and of adipose-specific insulin receptor deficiency (
      • Kershaw E.E
      • Hamm J.K.
      • Verhagen L.A.
      • Peroni O.
      • Katic M.
      • Flier J.S.
      Adipose triglyceride lipase: function, regulation by insulin, and comparison with adiponutrin..
      ). Both mouse models exhibited increased lipolysis and increased ATGL mRNA levels, indicating that the induction of ATGL gene expression might contribute to elevated FA mobilization under conditions of defective insulin signaling.
      Cytokines, and specifically TNFα, have multiple effects on adipose tissue, and TNFα has been implicated in the pathogenesis of obesity and insulin resistance (
      • Hotamisligil G.S
      Inflammation and metabolic disorders..
      ,
      • Wellen K.E
      • Hotamisligil G.S.
      Inflammation, stress, and diabetes..
      ). TNFα strongly stimulates lipolysis; as a consequence, massive lipid catabolism might contribute to the wasting seen in cancer cachexia. The finding that TNFα is able to stimulate lipolysis in HSL-ko adipocytes (
      • Okazaki H.
      • Osuga J.
      • Tamura Y.
      • Yahagi N.
      • Tomita S.
      • Shionoiri F.
      • Iizuka Y.
      • Ohashi K.
      • Harada K.
      • Kimura S.
      • et al.
      Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases..
      ) suggested that the process was HSL-independent and that ATGL could be the actual TNFα target lipase. However, although lipolysis is increased, two studies have reported that both ATGL and HSL mRNA levels decrease in 3T3-L1 adipocytes in response to TNFα treatment (
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Kralisch S.
      • Klein J.
      • Lossner U.
      • Bluher M.
      • Paschke R.
      • Stumvoll M.
      • Fasshauer M.
      Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes..
      ). This again suggests a dissociation of enzyme mRNA levels and enzyme activity. A possible explanation for low ATGL mRNA levels upon TNFα treatment relates to the fact that TNFα suppresses the expression of a large number of adipose-specific genes, leading to an “adipocyte dedifferentiation” process (
      • Ruan H.
      • Lodish H.F.
      Insulin resistance in adipose tissue: direct and indirect effects of tumor necrosis factor-alpha..
      ). One of these genes, peroxisome proliferator-activated receptor-γ (PPARγ), is a key nuclear receptor controlling adipocyte differentiation and metabolism (
      • Zhang B.
      • Berger J.
      • Hu E.
      • Szalkowski D.
      • White-Carrington S.
      • Spiegelman B.M.
      • Moller D.E.
      Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha..
      ). Kim et al. (
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ) demonstrated that ATGL is a direct transcriptional target gene for PPARγ, and PPARγ agonists such as rosiglitazone increase ATGL mRNA levels and induce lipolysis in various adipose models (
      • Kim J.Y
      • Tillison K.
      • Lee J.H.
      • Rearick D.A.
      • Smas C.M.
      The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3-L1 adipocytes and is a target for transactivation by PPARgamma..
      ,
      • Festuccia W.T
      • Laplante M.
      • Berthiaume M.
      • Gelinas Y.
      • Deshaies Y.
      PPARgamma agonism increases rat adipose tissue lipolysis, expression of glyceride lipases, and the response of lipolysis to hormonal control..
      ,
      • Kershaw E.E
      • Schupp M.
      • Guan H.P.
      • Gardner N.P.
      • Lazar M.A.
      • Flier J.S.
      PPARgamma regulates adipose triglyceride lipase in adipocytes in vitro and in vivo..
      ,
      • Kim J.Y
      • Wu Y.
      • Smas C.M.
      Characterization of ScAP-23, a new cell line from murine subcutaneous adipose tissue, identifies genes for the molecular definition of preadipocytes..
      ,
      • Shen W.J
      • Patel S.
      • Yu Z.
      • Jue D.
      • Kraemer F.B.
      Effects of rosiglitazone and high fat diet on lipase/esterase expression in adipose tissue..
      ). Therefore, it is conceivable that the TNFα-mediated inhibition of PPARγ reduces ATGL mRNA expression. How TNFα affects ATGL enzyme activity is currently unknown. In macrophages, ATGL has also been shown to be a target of PPARδ (
      • Lee C.H
      • Kang K.
      • Mehl I.R.
      • Nofsinger R.
      • Alaynick W.A.
      • Chong L.W.
      • Rosenfeld J.M.
      • Evans R.M.
      Peroxisome proliferator-activated receptor delta promotes very low-density lipoprotein-derived fatty acid catabolism in the macrophage..
      ).
      Contradicting views currently exist regarding the relative importance of ATGL in relation to HSL in human WAT. Langin (
      • Langin D.
      Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome..
      ) concluded from their studies in primary human adipocytes that HSL is the major lipase for catecholamine- and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates TG hydrolysis mainly during basal lipolysis. Another study suggested that human HSL has a higher capacity to hydrolyze TG compared with ATGL (
      • Mairal A.
      • Langin D.
      • Arner P.
      • Hoffstedt J.
      Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity..
      ). This study and a report by Ryden et al. (
      • Ryden M.
      • Jocken J.
      • van Harmelen V.
      • Dicker A.
      • Hoffstedt J.
      • Wiren M.
      • Blomqvist L.
      • Mairal A.
      • Langin D.
      • Blaak E.
      • et al.
      Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis..
      ) also found that, in contrast to HSL, ATGL mRNA and protein levels in adipose tissue are unaffected by obesity and weight reduction, arguing for a regulation of HSL, but not ATGL gene expression in response to obesity status. In contrast, other reports assign a crucial role to ATGL for TG hydrolysis in human WAT and show decreased ATGL mRNA and protein levels in obese individuals with insulin resistance (
      • Jocken J.W
      • Langin D.
      • Smit E.
      • Saris W.H.
      • Valle C.
      • Hul G.B.
      • Holm C.
      • Arner P.
      • Blaak E.E.
      Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state..
      ,
      • Steinberg G.R
      • Kemp B.E.
      • Watt M.J.
      Adipocyte triglyceride lipase expression in human obesity..
      ,
      • Berndt J.
      • Kralisch S.
      • Kloting N.
      • Ruschke K.
      • Kern M.
      • Fasshauer M.
      • Schon M.R.
      • Stumvoll M.
      • Bluher M.
      Adipose triglyceride lipase gene expression in human visceral obesity..
      ). The availability of specimens from patients with ATGL deficiency might help to elucidate the functional role of ATGL in human WAT.

      Regulation of ATGL: lipid droplet proteins

      CGI-58

      Mammalian TG hydrolases that act on water/lipid interphases frequently require cofactors for full enzyme function. For example, pancreatic lipase forms a complex with a colipase, and lipoprotein lipase (LPL) acts in concert with apolipoprotein C-II (apoC-II). ApoC-II is present on the surface of the major substrates for LPL, the TG-rich lipoproteins VLDL and chylomicrons. By analogy, it was not totally surprising when a lipid droplet protein, CGI-58 or ABHD5, was found to activate ATGL (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ). In the presence of CGI-58, the TG hydrolase activity of mouse ATGL is induced approximately 20-fold. Human ATGL is also activated by CGI-58, although to a lesser degree (approximately 5-fold ATGL induction). Importantly, these findings provided a biochemical explanation for a human disorder. In 2001, Lefevre et al. (
      • Lefevre C.
      • Jobard F.
      • Caux F.
      • Bouadjar B.
      • Karaduman A.
      • Heilig R.
      • Lakhdar H.
      • Wollenberg A.
      • Verret J.L.
      • Weissenbach J.
      • et al.
      Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome..
      ) discovered that mutations in the gene for CGI-58 are causative for a lipid storage disorder designated “neutral lipid storage disease” or Chanarin Dorfman Syndrome (see below for discussion of human mutations). CGI-58 was originally identified as a homologous gene in an alignment of the human and the Caenorhabditis elegans genomes. Mouse CGI-58 is ubiquitously expressed, with the highest expression levels found in testis and adipose tissue (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ,
      • Subramanian V.
      • Rothenberg A.
      • Gomez C.
      • Cohen A.W.
      • Garcia A.
      • Bhattacharyya S.
      • Shapiro L.
      • Dolios G.
      • Wang R.
      • Lisanti M.P.
      • et al.
      Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes..
      ).
      CGI-58 is a 349 amino acid-long protein with a molecular mass of 40 kDa. As shown in Fig. 2, the protein belongs to the esterase/thioesterase/lipase subfamily of proteins structurally characterized by the presence of α/β hydrolase folds. In contrast to most other members of this family, the putative nucleophilic serine within the canonical esterase/lipase motif GXSXG is replaced by an asparagine in CGI-58 (
      • Lefevre C.
      • Jobard F.
      • Caux F.
      • Bouadjar B.
      • Karaduman A.
      • Heilig R.
      • Lakhdar H.
      • Wollenberg A.
      • Verret J.L.
      • Weissenbach J.
      • et al.
      Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome..
      ), effectively eliminating the possibility that CGI-58 functions as a lipase. The protein was shown to bind to lipid droplets by interaction with perilipin A in a hormone-dependent way (
      • Subramanian V.
      • Rothenberg A.
      • Gomez C.
      • Cohen A.W.
      • Garcia A.
      • Bhattacharyya S.
      • Shapiro L.
      • Dolios G.
      • Wang R.
      • Lisanti M.P.
      • et al.
      Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes..
      ,
      • Vallet-Erdtmann V.
      • Tavernier G.
      • Contreras J.A.
      • Mairal A.
      • Rieu C.
      • Touzalin A.M.
      • Holm C.
      • Jegou B.
      • Langin D.
      The testicular form of hormone-sensitive lipase HSLtes confers rescue of male infertility in HSL-deficient mice..
      ,
      • Yamaguchi T.
      • Omatsu N.
      • Matsushita S.
      • Osumi T.
      CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome..
      ). In nonstimulated adipocytes, CGI-58 is tightly associated with the lipid droplet, whereas upon β-adrenergic stimulation and concomitant phosphorylation of perilipin, CGI-58 dissociates and becomes cytosolic (
      • Subramanian V.
      • Rothenberg A.
      • Gomez C.
      • Cohen A.W.
      • Garcia A.
      • Bhattacharyya S.
      • Shapiro L.
      • Dolios G.
      • Wang R.
      • Lisanti M.P.
      • et al.
      Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes..
      ,
      • Yamaguchi T.
      • Omatsu N.
      • Matsushita S.
      • Osumi T.
      CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome..
      ). Reducing the cAMP levels of the cell reverses this dissociation process (
      • Subramanian V.
      • Rothenberg A.
      • Gomez C.
      • Cohen A.W.
      • Garcia A.
      • Bhattacharyya S.
      • Shapiro L.
      • Dolios G.
      • Wang R.
      • Lisanti M.P.
      • et al.
      Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes..
      ). Fluorescence resonance energy transfer and bimolecular fluorescence complementation experiments showed that CGI-58, once dissociated from perilipin, colocalizes in close proximity to ATGL (
      • Granneman J.G
      • Moore H.P.
      • Granneman R.L.
      • Greenberg A.S.
      • Obin M.S.
      • Zhu Z.
      Analysis of lipolytic protein trafficking and interactions in adipocytes..
      ), suggesting the involvement of CGI-58/ATGL interaction in stimulated lipolysis. CGI-58 is not involved in the vesicularization of lipid droplets during lipolysis (
      • Yamaguchi T.
      • Omatsu N.
      • Morimoto E.
      • Nakashima H.
      • Ueno K.
      • Tanaka T.
      • Satouchi K.
      • Hirose F.
      • Osumi T.
      CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation..
      ). In summary, these findings support the following scenario: In the basal state, when adipocytes are not hormonally stimulated, CGI-58 binds to perilipin A and is unable to activate ATGL. Following hormonal stimulation, perilipin is phosphorylated at several serine residues, including serine 517, whereupon CGI-58 dissociates from perilipin, interacts with ATGL, and activates TG hydrolysis. Whether phosphorylation of serine 517 in perilipin or phosphorylation of ATGL affects the respective CGI-58 binding directly is currently not known. Concomitant with ATGL activation, HSL translocates from the cytosol to the lipid droplet and efficiently hydrolyzes DG, the lipolytic product of ATGL.
      Figure thumbnail gr2
      Fig. 2Representation of the structural domains of human comparative gene identification-58 (CGI-58) α/β hydrolase domain-containing protein 5 (ABHD5) (CGI-58/ABHD5). The α/β hydrolase region and the asparagine residue replacing a serine within the consensus GXSXG of lipases and esterases are indicated. Additionally, mutations in CGI-58 associated with neutral lipid storage disease with ichthyosis are shown. Two mutations within splice acceptor consensus sequences cause protein truncations after exon 2 and exon 5. The final amino acids of the wild-type sequences are indicated (K43SM and P256SM).
      Activation of ATGL might not be the only physiological activity of CGI-58. Importantly, a very recent publication showed that in addition to its function as ATGL activator, CGI-58 can also act as acylglycerolphosphate acyltransferase (AGPAT) (
      • Ghosh A.K
      • Ramakrishnan G.
      • Chandramohan C.
      • Rajasekharan R.
      CGI-58, the causative gene for Chanarin-Dorfman Syndrome, mediates acylation of lysophosphatidic acid..
      ). The role of this reaction in vivo remains to be determined (see below for discussion of human mutations).

      PAT proteins

      The crucial role of perilipin in the ATGL/CGI-58-mediated hydrolysis of TG became evident in an elegant study by Miyoshi et al. (
      • Miyoshi H.
      • Perfield II, J.W
      • Souza S.C.
      • Shen W.J.
      • Zhang H.H.
      • Stancheva Z.S.
      • Kraemer F.B.
      • Obin M.S.
      • Greenberg A.S.
      Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes..
      ) showing that hormone-stimulated lipolysis depended on perilipin and ATGL. The authors demonstrated that perilipin phosphorylation of residue serine-517 is essential for ATGL-mediated lipolysis and represents a prerequisite for the function of subsequent lipase activity of HSL.
      Perilipin expression is confined to adipose tissue and steroidogenic tissues. Lipolysis of lipid droplet-associated TG is, however, required in many other tissues, including those that do not express perilipin, such as skeletal and cardiac muscle, or the liver. Accordingly, alternative mechanisms must exist to control TG hydrolysis by ATGL and HSL (and possibly other lipases) in nonadipose tissues. These mechanisms are not well understood. Recently, two studies addressed the questions of whether and how nonperilipin PAT proteins affect lipolysis and ATGL. Listenberger et al. (
      • Listenberger L.L
      • Ostermeyer-Fay A.G.
      • Goldberg E.B.
      • Brown W.J.
      • Brown D.A.
      Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover..
      ) demonstrated that ADRP controls ATGL access and TG lipolysis in HEK293 cells and other human cell lines. Bell et al. (
      • Bell M.
      • Wang H.
      • Chen H.
      • McLenithan J.C.
      • Gong D.W.
      • Yang R.Z.
      • Yu D.
      • Fried S.K.
      • Quon M.J.
      • Londos C.
      • et al.
      Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance..
      ) studied the role of various PAT proteins in TG catabolism in hepatocyte-like AML12 cells and found that reduced expression of ADRP and TIP47 caused increased ATGL localization to lipid droplets and increased lipolytic rates. These findings are consistent with a crucial regulatory role for lipid droplet scaffold protein regulating the substrate access of functional ATGL.

      Pigment epithelium-derived factor

      In addition to PAT proteins, other proteins found on lipid droplets are also involved in the regulation of lipolysis. Surprisingly, searching for receptors and binding proteins for pigment epithelium-derived factor (PEDF), Notari et al. (
      • Notari L.
      • Baladron V.
      • Aroca-Aguilar J.D.
      • Balko N.
      • Heredia R.
      • Meyer C.
      • Notario P.M.
      • Saravanamuthu S.
      • Nueda M.L.
      • Sanchez-Sanchez F.
      • et al.
      Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor..
      ) identified ATGL as a PEDF binding protein and proposed to name the enzyme PEDF-receptor. Apparently, ATGL is highly expressed in the pigment epithelium and can be found on the plasma membrane, where it binds to PEDF and exhibits phospholipase activity. PEDF binding might also be important in cells and organs where ATGL is localized only within cells. For example, hepatocytes that lack PEDF were shown to accrue neutral lipid droplets, and lipid accumulation was reversed by the reexpression of PEDF (
      • Chung C.
      • Doll J.A.
      • Gattu A.K.
      • Shugrue C.
      • Cornwell M.
      • Fitchev P.
      • Crawford S.E.
      Anti-angiogenic pigment epithelium-derived factor regulates hepatocyte triglyceride content through adipose triglyceride lipase (ATGL)..
      ). These results suggest that PEDF binds to ATGL on lipid droplets, inducing TG hydrolysis. The mechanism of this activation and the question of whether PEDF mediates the activation of ATGL also in other tissues remain to be determined.

      CideN

      Another group of lipid droplet binding proteins that regulate lipolysis belongs to the CideN family. CideN proteins were originally discovered because of their structural similarity to DNA fragmentation factors and were believed to regulate cell death activation (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor..
      ). Recently, members of the CideN family were shown to affect lipid droplet morphology and turnover. CideA and CideC/Fsp27 bind to lipid droplets and colocalize with perilipin (
      • Puri V.
      • Konda S.
      • Ranjit S.
      • Aouadi M.
      • Chawla A.
      • Chouinard M.
      • Chakladar A.
      • Czech M.P.
      Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage..
      ,
      • Puri V.
      • Ranjit S.
      • Konda S.
      • Nicoloro S.M.
      • Straubhaar J.
      • Chawla A.
      • Chouinard M.
      • Lin C.
      • Burkart A.
      • Corvera S.
      • et al.
      Cidea is associated with lipid droplets and insulin sensitivity in humans..
      ). Overexpression of these factors inhibits fat catabolism and induces cellular lipid accumulation (
      • Puri V.
      • Konda S.
      • Ranjit S.
      • Aouadi M.
      • Chawla A.
      • Chouinard M.
      • Chakladar A.
      • Czech M.P.
      Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage..
      ). Consistent with these findings, mice that lack CideC/FSP27 have smaller, multilocular lipid droplets, decreased fat mass, lower levels of plasma FAs, and increased insulin sensitivity (
      • Nishino N.
      • Tamori Y.
      • Tateya S.
      • Kawaguchi T.
      • Shibakusa T.
      • Mizunoya W.
      • Inoue K.
      • Kitazawa R.
      • Kitazawa S.
      • Matsuki Y.
      • et al.
      FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets..
      ,
      • Toh S.Y
      • Gong J.
      • Du G.
      • Li J.Z.
      • Yang S.
      • Ye J.
      • Yao H.
      • Zhang Y.
      • Xue B.
      • Li Q.
      • et al.
      Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice..
      ). Similarly, CideA and CideB deficiency in mice is associated with a lean phenotype (
      • Li J.Z
      • Ye J.
      • Xue B.
      • Qi J.
      • Zhang J.
      • Zhou Z.
      • Li Q.
      • Wen Z.
      • Li P.
      Cideb regulates diet-induced obesity, liver steatosis, and insulin sensitivity by controlling lipogenesis and fatty acid oxidation..
      ,
      • Zhou Z.
      • Yon Toh S.
      • Chen Z.
      • Guo K.
      • Ng C.P.
      • Ponniah S.
      • Lin S.C.
      • Hong W.
      • Li P.
      Cidea-deficient mice have lean phenotype and are resistant to obesity..
      ). CideC/Fsp27 is also important for the regulation of TG catabolism in hepatocytes, because increased protein expression in ob/ob mice or in animals infected with CideC-expressing adenovirus causes hepatic lipid accumulation and steatosis (
      • Matsusue K.
      • Kusakabe T.
      • Noguchi T.
      • Takiguchi S.
      • Suzuki T.
      • Yamano S.
      • Gonzalez F.J.
      Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27..
      ). The mechanism by which members of the CideN family regulate the activity of lipases is currently unknown.

      Arf1-CopI

      In a genome-wide RNA interference screen in Drosophila S2 cells, Guo et al. (
      • Guo Y.
      • Walther T.C.
      • Rao M.
      • Stuurman N.
      • Goshima G.
      • Terayama K.
      • Wong J.S.
      • Vale R.D.
      • Walter P.
      • Farese R.V.
      Functional genomic screen reveals genes involved in lipid-droplet formation and utilization..
      ) identified a large number of genes that affect lipid droplet biogenesis and morphology. Interestingly, the study identified a subset of the Arf1-CopI family of vesicular transport proteins that strongly affect lipid mobilization. Silencing of Arf79F or CopI resulted in smaller, more disperse lipid droplets and increased lipolysis, suggesting yet another currently unknown mechanism that regulates the activity of lipolytic enzymes.
      Taken together, these results suggest that lipases are embedded in a complex “lipolysome” consisting of the actual lipolytic enzymes and numerous modulators of enzyme activity.

      MUTATIONS IN ATGL OR CGI-58 CAUSE NEUTRAL LIPID STORAGE DISEASE IN HUMANS

      Recently, mutations in the genes for ATGL and CGI-58 were identified and provided the molecular basis underlying neutral lipid storage disease (NLSD) in humans. NLSD is a rare, autosomal genetic disorder characterized by systemic accumulation of TG in all tissues of the body. It is diagnosed by increased TG storage in blood granulocytes (referred to as Jordans' anomaly) (
      • Pena-Penabad C.
      • Almagro M.
      • Martinez W.
      • Garcia-Silva J.
      • Del Pozo J.
      • Yebra M.T.
      • Sanchez-Manzano C.
      • Fonseca E.
      Dorfman–Chanarin syndrome (neutral lipid storage disease): new clinical features..
      ). Excessive lipid storage leads to variable forms of skeletal and cardiac myopathy and hepatic steatosis. Additionally, some patients suffer from ataxia, hearing loss, or mental retardation (
      • Chanarin I.
      • Patel A.
      • Slavin G.
      • Wills E.J.
      • Andrews T.M.
      • Stewart G.
      Neutral-lipid storage disease: a new disorder of lipid metabolism..
      ,
      • Dorfman M.L
      • Hershko C.
      • Eisenberg S.
      • Sagher F.
      Ichthyosiform dermatosis with systemic lipidosis..
      ). According to a recently proposed classification (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ), NLSD can be subdivided into two distinct groups. Depending on whether or not the patients suffer from a skin defect (severe ichthyosis), they are diagnosed with either neutral lipid storage disease with ichthyosis (NLSDI, also known as Chanarin Dorfman Syndrome) or neutral lipid storage disease with myopathy (NLSDM), respectively. Importantly, this classification finds its molecular basis in the affected genes. Mutations in the gene for ATGL (PNPLA2) cause NLSDM, and mutations in the gene for CGI-58 cause NLSDI.

      ATGL

      In 2007, Fischer et al. (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ) reported that mutations in the gene for ATGL cause NLSDM. Since then, several new mutations in the ATGL gene locus (PNPLA2) were discovered (
      • Akiyama M.
      • Sakai K.
      • Ogawa M.
      • McMillan J.R.
      • Sawamura D.
      • Shimizu H.
      Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy..
      ,
      • Kobayashi K.
      • Inoguchi T.
      • Maeda Y.
      • Nakashima N.
      • Kuwano A.
      • Eto E.
      • Ueno N.
      • Sasaki S.
      • Sawada F.
      • Fujii M.
      • et al.
      The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets..
      ). Currently, six mutations are known to cause aberrant ATGL proteins (indicated in Fig. 1). These include a point mutation (Pro195Leu), four frameshift mutations (at amino acids 160, 267, 270, or 283), and one nonsense mutation (Asn289×). Both frameshift and nonsense mutations result in the deletion of the C-terminal region of ATGL. Interestingly, the patatin domain with the active site serine 47/aspartate 166 dyad is present in most of the truncated ATGL variants. These ATGL mutants are enzymatically highly active and can be stimulated by CGI-58 when artificial lipid emulsions are used as substrates (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ,
      • Kobayashi K.
      • Inoguchi T.
      • Maeda Y.
      • Nakashima N.
      • Kuwano A.
      • Eto E.
      • Ueno N.
      • Sasaki S.
      • Sawada F.
      • Fujii M.
      • et al.
      The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets..
      ,
      • Schweiger M.
      • Schoiswohl G.
      • Lass A.
      • Radner F.P.
      • Haemmerle G.
      • Malli R.
      • Graier W.
      • Cornaciu I.
      • Oberer M.
      • Salvayre R.
      • et al.
      The C-terminal region of human adipose triglyceride lipase affects enzyme activity and lipid droplet binding..
      ). However, ATGL lacking parts of the C-terminal half of the enzyme exhibited reduced binding to cellular lipid droplets, and it is assumed that defective substrate binding in a cellular context is responsible for the lipolytic defect. In one patient with severe myopathy, the mutation occurred within the patatin domain (frameshift mutation at amino acid 160); this mutation results in a truncated protein that lacks the active site aspartate 166 (
      • Akiyama M.
      • Sakai K.
      • Ogawa M.
      • McMillan J.R.
      • Sawamura D.
      • Shimizu H.
      Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy..
      ). Whether this mutation results in complete enzyme inactivation is not known. True null mutants that totally lack ATGL have not been found so far. More extensive biochemical characterization of ATGL deletion mutants retaining the patatin and α/β hydrolase domain but lacking the C-terminal region revealed a double function of the C terminus (
      • Schweiger M.
      • Schoiswohl G.
      • Lass A.
      • Radner F.P.
      • Haemmerle G.
      • Malli R.
      • Graier W.
      • Cornaciu I.
      • Oberer M.
      • Salvayre R.
      • et al.
      The C-terminal region of human adipose triglyceride lipase affects enzyme activity and lipid droplet binding..
      ). First, it appears to mediate lipid droplet binding. Second, it has an inhibitory role in the hydrolytic reaction, because absence of the C-terminal region generates an enzyme with higher specific activity against artificial substrates. Whether the established phosphorylation sites present in this region (
      • Bartz R.
      • Zehmer J.K.
      • Zhu M.
      • Chen Y.
      • Serrero G.
      • Zhao Y.
      • Liu P.
      Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation..
      ) affect lipid binding or enzyme activity is presently unknown.
      The clinical observations in patients with NLSDM resemble in many aspects the phenotypic characteristics of ATGL-ko mice. Both genetic deficiencies result in systemic lipid accumulation, Jordans' anomaly, and myopathy. Skin defects were not observed in either species. Two patients with NLSDM were reported to have died from cardiac failure (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ,
      • Kobayashi K.
      • Inoguchi T.
      • Maeda Y.
      • Nakashima N.
      • Kuwano A.
      • Eto E.
      • Ueno N.
      • Sasaki S.
      • Sawada F.
      • Fujii M.
      • et al.
      The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets..
      ), but unfortunately it is not known whether their cardiac dysfunction resulted from excessive lipid accumulation as observed in mice. The identification and characterization of additional patients with PNPLA2 gene mutations will help to elucidate the role of ATGL in human cardiac physiology.

      CGI-58

      Six years before mutations in ATGL were found to cause NLSDM, Lefèvre et al. (
      • Lefevre C.
      • Jobard F.
      • Caux F.
      • Bouadjar B.
      • Karaduman A.
      • Heilig R.
      • Lakhdar H.
      • Wollenberg A.
      • Verret J.L.
      • Weissenbach J.
      • et al.
      Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome..
      ) described eight mutations in the human gene for CGI-58 in families with a confirmed diagnosis of NLSDI. Subsequently, other groups reported additional mutations in the gene for CGI-58 causative for NLSDI (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ,
      • Akiyama M.
      • Sawamura D.
      • Nomura Y.
      • Sugawara M.
      • Shimizu H.
      Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome..
      ,
      • Ben Selma Z.
      • Yilmaz S.
      • Schischmanoff P.O.
      • Blom A.
      • Ozogul C.
      • Laroche L.
      • Caux F.
      A novel S115G mutation of CGI-58 in a Turkish patient with Dorfman-Chanarin syndrome..
      ). The locations of the known mutations in the CGI-58 sequence that cause NLSDI are included in Fig. 2. Patients share many clinical features with those affected with ATGL deficiency, but some striking differences are apparent. All patients with CGI-58 deficiency suffer from severe ichthyosis, and some of them have developmental defects, including deformation of the ear and mental retardation. These differences suggest that CGI-58 has additional functions that are independent of ATGL. Mutated CGI-58 with single amino acid substitutions (Q130P, E260K) or deletions totally fail to activate ATGL (
      • Lass A.
      • Zimmermann R.
      • Haemmerle G.
      • Riederer M.
      • Schoiswohl G.
      • Schweiger M.
      • Kienesberger P.
      • Strauss J.G.
      • Gorkiewicz G.
      • Zechner R.
      Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome..
      ), suggesting that the defective ATGL stimulation by CGI-58 is causative for the multi-tissue TG accumulation observed in NLSDI. Interestingly, the same mutations in CGI-58 are unable to bind to perilipin and are not associated with lipid droplets (
      • Yamaguchi T.
      • Omatsu N.
      • Matsushita S.
      • Osumi T.
      CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome..
      ). Alternative explanations for the molecular defect in NLSDI have also been proposed. Even before CGI-58 was discovered, it was shown that the molecular defect present in NLSDI prevents lipid remodeling from neutral lipids to glycerophospholipids (
      • Igal R.A
      • Coleman R.A.
      Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid storage disease fibroblasts..
      ,
      • Williams M.L
      • Coleman R.A.
      • Placezk D.
      • Grunfeld C.
      Neutral lipid storage disease: a possible functional defect in phospholipid-linked triacylglycerol metabolism..
      ). Considering the recent finding that CGI-58 exhibits AGPAT activity (
      • Ghosh A.K
      • Ramakrishnan G.
      • Chandramohan C.
      • Rajasekharan R.
      CGI-58, the causative gene for Chanarin-Dorfman Syndrome, mediates acylation of lysophosphatidic acid..
      ) and might affect phospholipid synthesis, it seems conceivable that both defective TG hydrolysis and phospholipid synthesis contribute to the pathogenesis of NLSDI. However, several issues need clarification. First, the finding that mutations in CGI-58 causing NLSDI had no effect on its activity as acyltransferase raises the question of whether this activity is lacking in patients with NLSDI. Second, how does a defect in AGPAT activity of CGI-58 cause massive TG accumulation in light of the fact that the product of the AGPAT reaction, phosphatidic acid, is a common precursor for both TG and glycerophospholipids? Normally, AGPAT deficiency results in lipodystrophy and not excessive lipid accumulation (
      • Agarwal A.K
      • Garg A.
      Congenital generalized lipodystrophy: significance of triglyceride biosynthetic pathways..
      ).
      Taken together, the clinical phenotype of patients affected with both forms of NLSD and the comparison to the phenotype of ATGL-ko mice suggest that excessive lipid accumulation results from decreased lipolysis due to defects in the enzyme (ATGL) or its activator (CGI-58). In the skin, however, CGI-58 has an additional, ATGL-independent function that is defective in NLSDI and responsible for the development of ichthyosis. Whether this function of CGI-58 involves the activation of another lipase, alterations in the metabolism of phospholipids, or a completely unrelated activity remains to be determined.

      THE TISSUE-SPECIFIC ROLE OF ATGL/CGI-58

      With the availability of genetically modified mice that lack ATGL (CGI-ko mice have not been reported to date) and the characterization of patients with NLSDM and NLSDI, a picture emerges of how ATGL affects lipid metabolism and energy homeostasis. As a general conclusion, it is evident that the physiological function of ATGL/CGI-58 is not restricted to adipose tissue but is also crucially important in many nonadipose tissues.

      WAT

      ATGL deficiency in mice is associated with a major defect in WAT lipolysis (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ). Both TG hydrolase activity in WAT lysates and the release of FAs and glycerol from intact tissue samples are markedly increased. As a consequence, 8-week-old ATGL-deficient mice are obese, exhibiting double the fat mass of normal mice when kept on a normal chow diet. In contrast to mice, human patients with ATGL or CGI-58 deficiency are not overweight or obese. This has been used as argument that ATGL-mediated lipolysis in human WAT is less important than in mouse WAT. However, other explanations are also conceivable. First, defective lipolysis in WAT can result in a concomitant downregulation of lipogenesis. For example, the loss of WAT mass in HSL-deficient mice is caused by a drastic reduction of lipogenesis due to decreased PPARγ activity (
      • Zimmermann R.
      • Haemmerle G.
      • Wagner E.M.
      • Strauss J.G.
      • Kratky D.
      • Zechner R.
      Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue..
      ). Second, patients might change their eating habits as a consequence of their disease. Third, the absence of ATGL in human WAT might induce alternative lipase activities. The analysis of tissue samples from patients suffering from NLSDM or NLSDI will hopefully help to assess the role of ATGL in human WAT.

      BAT

      BAT serves as a TG storage organ with the unique ability to generate heat by “non-shivering” thermogenesis. In humans, brown adipocytes are abundant in neonates and diminish with age. In rodents and hibernating animals, BAT persists throughout life and is a major site for heat production in response to low environmental temperature. Similarly to the situation in WAT, β-adrenergic stimulation in BAT promotes the hydrolysis of stored TG by endogenous lipases, leading to the mobilization of FAs as fuel for thermogenesis. Defective norepinephrine and epinephrine synthesis (
      • Thomas S.A
      • Palmiter R.D.
      Thermoregulatory and metabolic phenotypes of mice lacking noradrenaline and adrenaline..
      ) or deficiency of all three known β-adrenergic receptors (
      • Bachman E.S
      • Dhillon H.
      • Zhang C.Y.
      • Cinti S.
      • Bianco A.C.
      • Kobilka B.K.
      • Lowell B.B.
      betaAR signaling required for diet-induced thermogenesis and obesity resistance..
      ) results in reduced lipolysis, increased BAT mass, and severe cold sensitivity due to defective thermogenesis. Remarkably, mice lacking HSL exhibited normal thermogenesis (
      • Osuga J.
      • Ishibashi S.
      • Oka T.
      • Yagyu H.
      • Tozawa R.
      • Fujimoto A.
      • Shionoiri F.
      • Yahagi N.
      • Kraemer F.B.
      • Tsutsumi O.
      • et al.
      Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity..
      ) and were not cold sensitive despite a lipolytic defect that resulted in brown adipocyte hypertrophy due to TG and DG accumulation. Apparently, in the absence of HSL, sufficient amounts of FAs are mobilized for mitochondrial heat production. In contrast, ATGL-ko mice are extremely cold sensitive and die after cold exposure of more than 6 h, indicating that the enzyme is essential for the provision of FA as substrate for thermogenesis (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ).

      Cardiac muscle

      In the heart, continuous energy production is indispensable for the supply of ATP required for the permanent contractile function of the beating heart. It is estimated that 50–70% of the energy for myocardial contraction derives from the oxidation of FAs (
      • van der Vusse G.J
      • van Bilsen M.
      • Glatz J.F
      Cardiac fatty acid uptake and transport in health and disease..
      ). Because cardiomyocytes do not synthesize FAs, they depend on their supply from two exogenous sources: first, WAT-derived, circulating unesterified FAs that are bound to plasma albumin; and second, TG-associated FAs released by LPL from TG-rich plasma lipoproteins. LPL is the only enzyme known to be responsible for the hydrolysis of plasma TG-rich lipoproteins in peripheral cells (
      • Goldberg I.J
      Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis..
      ,
      • Preiss-Landl K.
      • Zimmermann R.
      • Hammerle G.
      • Zechner R.
      Lipoprotein lipase: the regulation of tissue specific expression and its role in lipid and energy metabolism..
      ). Experiments in transgenic and ko mouse models showed that the expression level of LPL in the heart largely determines the uptake rate of FA. Increased LPL activity in the heart result in elevated FA utilization, increased lipid storage, decreased glucose utilization, and modest signs of cardiomyopathy (
      • Augustus A.
      • Yagyu H.
      • Haemmerle G.
      • Bensadoun A.
      • Vikramadithyan R.K.
      • Park S.Y.
      • Kim J.K.
      • Zechner R.
      • Goldberg I.J.
      Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression..
      ,
      • Levak-Frank S.
      • Hofmann W.
      • Weinstock P.H.
      • Radner H.
      • Sattler W.
      • Breslow J.L.
      • Zechner R.
      Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels..
      ,
      • Noh H.L
      • Okajima K.
      • Molkentin J.D.
      • Homma S.
      • Goldberg I.J.
      Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction..
      ,
      • Augustus A.S
      • Buchanan J.
      • Park T.S.
      • Hirata K.
      • Noh H.L.
      • Sun J.
      • Homma S.
      • D'Armiento J.
      • Abel E.D.
      • Goldberg I.J.
      Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction..
      ,
      • Pillutla P.
      • Hwang Y.C.
      • Augustus A.
      • Yokoyama M.
      • Yagyu H.
      • Johnston T.P.
      • Kaneko M.
      • Ramasamy R.
      • Goldberg I.J.
      Perfusion of hearts with triglyceride-rich particles reproduces the metabolic abnormalities in lipotoxic cardiomyopathy..
      ,
      • Yagyu H.
      • Chen G.
      • Yokoyama M.
      • Hirata K.
      • Augustus A.
      • Kako Y.
      • Seo T.
      • Hu Y.
      • Lutz E.P.
      • Merkel M.
      • et al.
      Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy..
      ). These studies and previous investigations showed that FAs, once absorbed by the heart, are not utilized directly for β-oxidation but, at least in part, are converted into TG (
      • Swanton E.M
      • Saggerson E.D.
      Effects of adrenaline on triacylglycerol synthesis and turnover in ventricular myocytes from adult rats..
      ). Subsequently, intracellular lipase(s) hydrolyze(s) these TG depots and provide(s) FA for oxidation. Accordingly, endogenous myocardial TGs provide a substantial amount of FAs for oxidation in perfused rat hearts, especially under conditions of overt diabetes (
      • Saddik M.
      • Lopaschuk G.D.
      Triacylglycerol turnover in isolated working hearts of acutely diabetic rats..
      ). When hydrolyzed FAs are not utilized for oxidation, they are again esterified to TG. This creates a futile cycle of lipolysis and reesterification that reacts promptly to meet increased substrate demand when energy is needed.
      Cardiomyocytes express HSL, and the hormone responsiveness of myocardial lipolysis suggested that HSL might be sufficient for TG hydrolysis in the heart (
      • Holm C.
      • Belfrage P.
      • Fredrikson G.
      Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue..
      ,
      • Small C.A
      • Garton A.J.
      • Yeaman S.J.
      The presence and role of hormone-sensitive lipase in heart muscle..
      ). However, HSL-deficient mice do not accumulate TG in the heart (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ). In fact, overnight-fasted HSL-ko mice show markedly reduced myocardial TG levels and increased LPL activity (
      • Haemmerle G.
      • Zimmermann R.
      • Strauss J.G.
      • Kratky D.
      • Riederer M.
      • Knipping G.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle..
      ), suggesting that both uptake of lipoprotein-associated FAs and intramyocardial TG mobilization are still functional when HSL is absent. ATGL-ko mice, in contrast, exhibit a prominent heart phenotype (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ). As early as 6 weeks after birth, ATGL-deficient hearts accumulate lipids, as evident by increased number and size of lipid droplets; a process resulting in massive TG accumulation and yellowish discoloration of the heart. Lipid accumulation leads to an increased heart mass, decreased contractility, severe cardiac insufficiency, and premature death starting at about 12 weeks after birth. Remarkably, cardiac LPL activity is upregulated, indicating that despite massive TG accumulation, the FA uptake machinery is maximally induced. However, due to low VLDL levels in plasma during fasting, elevated LPL activities are probably not associated with increased FA uptake in cardiomyocytes. Decreased FA absorption from plasma is counterbalanced by increased uptake of glucose in ATGL-ko hearts. Thus, in the absence of ATGL, the release of FA from TG is blocked, leading to massive TG accumulation, decreased FA oxidation, and increased glucose utilization for energy production.
      A recently discovered pathway to reduce excessive cardiac lipids involves the synthesis and secretion of apoB-containing lipoproteins (
      • Nielsen L.B
      • Veniant M.
      • Boren J.
      • Raabe M.
      • Wong J.S.
      • Tam C.
      • Flynn L.
      • Vanni-Reyes T.
      • Gunn M.D.
      • Goldberg I.J.
      • et al.
      Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins..
      ). This mobilization of myocardial TG for lipoprotein secretion is thought to provide the heart with a “safety valve” for the disposal of excess lipids. It has been proposed that this process requires the hydrolysis of cytoplasmic TG stores and resynthesis of TG in the endoplamic reticulum. Whether this lipolytic step requires ATGL is presently unknown. Indirect evidence for such an involvement is provided by the fact that cardiac lipoprotein synthesis apparently cannot prevent the lethal lipid accumulation in hearts of ATGL-ko mice.
      Whether defective ATGL function in humans with NLSDM or NLSDI also causes cardiac dysfunction is not clear from the few cases known so far. Cardiomyopathy was reported in patients with both conditions, NLSDM (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ,
      • Akiyama M.
      • Sakai K.
      • Ogawa M.
      • McMillan J.R.
      • Sawamura D.
      • Shimizu H.
      Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy..
      ) and NLSDI (
      • Igal R.A
      • Rhoads J.M.
      • Coleman R.A.
      Neutral lipid storage disease with fatty liver and cholestasis..
      ), although it appears to be much less severe or less frequent in the latter. Reportedly, two patients with ATGL deficiency died from cardiac failure, but it is not known whether excessive lipid accumulation caused the premature death.

      Skeletal muscle

      The release of FA from TG within skeletal myofibrillar lipid droplets requires lipases (
      • Blaak E.E
      Metabolic fluxes in skeletal muscle in relation to obesity and insulin resistance..
      ). The presence of HSL mRNA, protein, and enzyme activity has been documented in rodent (
      • Holm C.
      • Belfrage P.
      • Fredrikson G.
      Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue..
      ,
      • Langfort J.
      • Ploug T.
      • Ihlemann J.
      • Saldo M.
      • Holm C.
      • Galbo H.
      Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle..
      ,
      • Peters S.J
      • Dyck D.J.
      • Bonen A.
      • Spriet L.L.
      Effects of epinephrine on lipid metabolism in resting skeletal muscle..
      ) and in human skeletal muscle (
      • Roepstorff C.
      • Vistisen B.
      • Donsmark M.
      • Nielsen J.N.
      • Galbo H.
      • Green K.A.
      • Hardie D.G.
      • Wojtaszewski J.F.
      • Richter E.A.
      • Kiens B.
      Regulation of hormone-sensitive lipase activity and Ser563 and Ser565 phosphorylation in human skeletal muscle during exercise..
      ) by several laboratories. Compatible with its physiological function, HSL expression in skeletal muscle varies between fiber types, being higher in oxidative than in glycolytic fibers (
      • Langfort J.
      • Ploug T.
      • Ihlemann J.
      • Saldo M.
      • Holm C.
      • Galbo H.
      Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle..
      ,
      • Peters S.J
      • Dyck D.J.
      • Bonen A.
      • Spriet L.L.
      Effects of epinephrine on lipid metabolism in resting skeletal muscle..
      ). HSL in skeletal muscle is activated by PKA-mediated phosphorylation, a contraction-induced mechanism involving PKC, and the ERK pathway (
      • Langfort J.
      • Ploug T.
      • Ihlemann J.
      • Saldo M.
      • Holm C.
      • Galbo H.
      Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle..
      ,
      • Donsmark M.
      • Langfort J.
      • Holm C.
      • Ploug T.
      • Galbo H.
      Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase..
      ,
      • Langfort J.
      • Ploug T.
      • Ihlemann J.
      • Holm C.
      • Galbo H.
      Stimulation of hormone-sensitive lipase activity by contractions in rat skeletal muscle..
      ). An inhibitory effect of AMPK on HSL activity in resting and contracting muscle was reported by some (
      • Muoio D.M
      • Seefeld K.
      • Witters L.A.
      • Coleman R.A.
      AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target..
      ,
      • Smith A.C
      • Bruce C.R.
      • Dyck D.J.
      AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus muscle..
      ,
      • Watt M.J
      • Steinberg G.R.
      • Chan S.
      • Garnham A.
      • Kemp B.E.
      • Febbraio M.A.
      Beta-adrenergic stimulation of skeletal muscle HSL can be overridden by AMPK signaling..
      ) but not all studies (
      • Roepstorff C.
      • Vistisen B.
      • Donsmark M.
      • Nielsen J.N.
      • Galbo H.
      • Green K.A.
      • Hardie D.G.
      • Wojtaszewski J.F.
      • Richter E.A.
      • Kiens B.
      Regulation of hormone-sensitive lipase activity and Ser563 and Ser565 phosphorylation in human skeletal muscle during exercise..
      ). Despite the established role of HSL for the hydrolysis of stored TG in skeletal muscle, the absence of the enzyme in skeletal muscle of HSL-ko mice did not result in elevated muscular TG content or defects in muscle function (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis..
      ,
      • Osuga J.
      • Ishibashi S.
      • Oka T.
      • Yagyu H.
      • Tozawa R.
      • Fujimoto A.
      • Shionoiri F.
      • Yahagi N.
      • Kraemer F.B.
      • Tsutsumi O.
      • et al.
      Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity..
      ,
      • Wang S.P
      • Laurin N.
      • Himms-Hagen J.
      • Rudnicki M.A.
      • Levy E.
      • Robert M.F.
      • Pan L.
      • Oligny L.
      • Mitchell G.A.
      The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice..
      ). Instead, similarly to that observed in other tissues, HSL deficiency led to increased DG levels in skeletal muscle. Expression studies of genes involved in energy metabolism revealed that enzymes involved in carbohydrate metabolism are upregulated in HSL-ko skeletal muscles, whereas enzymes involved in FA biosynthesis are downregulated (
      • Pinent M.
      • Hackl H.
      • Burkard T.R.
      • Prokesch A.
      • Papak C.
      • Scheideler M.
      • Hammerle G.
      • Zechner R.
      • Trajanoski Z.
      • Strauss J.G.
      Differential transcriptional modulation of biological processes in adipocyte triglyceride lipase and hormone-sensitive lipase-deficient mice..
      ,
      • Hansson O.
      • Donsmark M.
      • Ling C.
      • Nevsten P.
      • Danfelter M.
      • Andersen J.L.
      • Galbo H.
      • Holm C.
      Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice..
      ).
      In contrast to HSL-ko mice, ATGL-ko mice accumulated TG in skeletal muscle (
      • Haemmerle G.
      • Lass A.
      • Zimmermann R.
      • Gorkiewicz G.
      • Meyer C.
      • Rozman J.
      • Heldmaier G.
      • Maier R.
      • Theussl C.
      • Eder S.
      • et al.
      Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase..
      ), supporting the concept that in addition to HSL, ATGL is also involved in the lipolytic cascade in myocytes. The absence of ATGL in skeletal muscle causes reduced lipolytic activity, neutral lipid droplet accumulation in oxidative muscle fibers, and elevated glucose uptake. Increased respiratory quotient (RQ) values during fasting in ATGL-ko compared with wild-type animals indicated increased glucose utilization in the absence of ATGL. Additional evidence for a functional role of ATGL was provided by Watt et al. (
      • Watt M.J
      • van Denderen B.J
      • Castelli L.A.
      • Bruce C.R.
      • Hoy A.J.
      • Kraegen E.W.
      • Macaulay L.
      • Kemp B.E.
      Adipose triglyceride lipase regulation of skeletal muscle lipid metabolism and insulin responsiveness..
      ), showing that overexpression of ATGL in skleletal muscle increases the oxidation of FA from TG stores and increases DG and ceramide production. Apparently, in this experimental setup, the endogenous HSL activity was not sufficient to hydrolyze excess DG. Accordingly, a dysequilibrium between the activities for ATGL and HSL might contribute to the production of lipotoxic intermediates and promote insulin resistance.
      The crucial role of ATGL in skeletal muscle energy metabolism in humans is strikingly supported by recent findings in patients with NLSD. Individuals that lack ATGL (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ,
      • Akiyama M.
      • Sakai K.
      • Ogawa M.
      • McMillan J.R.
      • Sawamura D.
      • Shimizu H.
      Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy..
      ,
      • Kobayashi K.
      • Inoguchi T.
      • Maeda Y.
      • Nakashima N.
      • Kuwano A.
      • Eto E.
      • Ueno N.
      • Sasaki S.
      • Sawada F.
      • Fujii M.
      • et al.
      The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets..
      ,
      • Akiyama M.
      • Sawamura D.
      • Nomura Y.
      • Sugawara M.
      • Shimizu H.
      Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome..
      ) or its activator, CGI-58 (
      • Pena-Penabad C.
      • Almagro M.
      • Martinez W.
      • Garcia-Silva J.
      • Del Pozo J.
      • Yebra M.T.
      • Sanchez-Manzano C.
      • Fonseca E.
      Dorfman–Chanarin syndrome (neutral lipid storage disease): new clinical features..
      ), accumulate TG in myocytes and develop muscle weakness and skeletal myopathy. Myopathy appears to be consistently more severe in patients with defective ATGL (NLSDM). The clinical phenotype of complete ATGL deficiency is not known because patients that completely lack ATGL have not been found so far. However, the severe myopathy in a patient with a frameshift mutation at amino acid 160 suggests that patients lacking the patatin domain are affected with a more severe form of the disease than those with an intact patatin domain (
      • Fischer J.
      • Lefevre C.
      • Morava E.
      • Mussini J.M.
      • Laforet P.
      • Negre-Salvayre A.
      • Lathrop M.
      • Salvayre R.
      The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy..
      ,
      • Akiyama M.
      • Sakai K.
      • Ogawa M.
      • McMillan J.R.
      • Sawamura D.
      • Shimizu H.
      Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy..