Catecholamine-induced regulation

Catecholamines, adrenaline and noradrenaline, exert their impact on lipolysis upon binding to the diverse adrenergic receptor subtypes located on the plasma membrane of adipocytes⁽ Reference Arner ^{2 ,} Reference Arner ^{45 ,} Reference Jocken and Blaak ^{53 )}. These receptors are linked to G-proteins, with G-protein receptor complexes regulating adenylate cyclase in the cell membrane. In mammals at least four adrenoceptors exert their action with marked species characteristics⁽ Reference Lafontan and Langin ^{4 )}. In humans β₁- and β₂-adrenoceptors are the most active lipolytic elements, while the contribution of β₃-adrenergic receptors remains to be better established. The presence of β₃-adrenoceptors in human white adipocytes has been clearly proven with tissue and subcellular distribution as well as response to stimulators being consistent with participation in lipolysis⁽ Reference De Matteis, Arch and Petroni ^{54 )}. However, the failure of β₃-adrenoceptor agonists to elicit clear-cut lipolytic and weight-loss effects in obese patients casted doubts on the true physiological relevance of this β-adrenoceptor subtype in humans⁽ Reference Buemann, Toubro and Astrup ^{55 ,} Reference Redman, de Jonge and Fang ^{56 )}. Contrarily, β₃-adrenoceptors are abundantly expressed in adipocytes of rodents⁽ Reference Gómez-Ambrosi, Frühbeck and Aguado ^{57 )}. Upon binding to their ligand, β-adrenergic receptors initiate the activation of the lipolytic cascade through the stimulation of cAMP production and subsequent activation of the cAMP-dependent PKA, which is followed by the phosphorylation of perilipin and HSL, ultimately leading to lipolysis stimulation (Fig. 2). Another peculiarity of human adipocytes resides in the presence of abundant α₂-adrenoceptors, which are coupled to G-inhibitory proteins (G_i), thereby inhibiting cAMP production and, thus, lipolysis⁽ Reference Lafontan and Berlan ^{58 ,} Reference Langin ^{59 )}. Therefore, the balance between the lipolytic effect of β-adrenergic receptors and the opposing anti-lipolytic activity of α₂-adrenoceptors also determines the net outcome of catecholamine-induced fat mobilisation in humans. The identification of brown adipose tissue in human adults beyond the vestigial amounts originally acknowledged and its association with BMI and adiposity has triggered a re-focusing of attention to the true relevance of β₃-adrenoceptors in lipid metabolism and energy homeostasis⁽ Reference Frühbeck, Becerril and Sáinz ^{60 ,} Reference Frühbeck, Sesma and Burrell ^{61 )}.

Fig. 2 Principal regulators and major pathways involved in adipocyte lipolysis. A₁R, A₁ adenosine receptor; AC, adenylyl cyclase; ADRP, adipophilin/adipocyte differentiation-related protein; AMPK, AMP-activated protein kinase; AQP7, aquaporin 7; AR, adrenoreceptor; ATGL, adipocyte TAG lipase; cAMP, cyclic AMP; CGI-58, comparative gene identification-58; cGMP, cyclic GMP; CIDEA, cell death-inducing DFFA (DNA fragmentation factor-α)-like effector A; CL, calcitonin receptor-like; EP-3R, PGE receptor 3; FABP4, fatty acid binding protein 4; FSP27, fat-specific protein 27; GC, guanylyl cyclase; G_i, inhibitory GTP-binding proteins; G_s, stimulatory GTP-binding proteins; HSL, hormone-sensitive lipase; IRS-1, insulin receptor substrate-1; JNK, Jun kinase; NOS, NO synthase; NPR, natriuretic peptide receptor; NPY, neuropeptide Y; NPY-R1, neuropeptide Y receptor 1; PDE3B, phosphodiesterase 3B; PEDF, pigment epithelium-derived factor; PI3K, phospatidylinositol-3 kinase; PKA, protein kinase A; PKB, protein kinase B; PKG, protein kinase G; RAMP2, receptor activity modifying protein-2; TIP47, tail-interacting protein of 47 kDa; TNF-α-R, TNF-α receptor; ZAG, zinc-α₂-glycoprotein. (A colour version of this figure can be found online at http://www.journals.cambridge.org/nrr)

Hormone-mediated control

A number of hormones are known to participate in the regulation of lipolysis. Among all endocrine factors, insulin is quantitatively and qualitatively the most relevant one. The impact of growth hormone (GH), adrenocorticotropic hormone, cortisol, thyroid hormones, PTH and glucagon is comparatively much more reduced than that of insulin. The mechanisms of action of all are briefly discussed below.

Hormone-mediated control: insulin

Insulin is a key regulator of white adipose tissue biology, controlling not only lipogenesis but also the rate of lipolysis and NEFA efflux. Insulin regulates glucose uptake by adipocytes and triggers fatty acid transport protein translocation as well as fatty acid uptake by fat cells⁽ Reference Lafontan ^{62 )}. Binding of insulin to its specific cell-surface receptor produces tyrosine phosphorylation and activation of the insulin receptor, which leads to the interaction with the insulin receptor substrates (IRS-1 and IRS-2), in turn activating the phosphatidyl inositol 3-kinase (PI3K) complex⁽ Reference Arner ^{2 )}. Insulin powerfully inhibits basal and catecholamine-induced lipolysis through phosphorylation (via a PKB/Akt-dependent action) and activation of phosphodiesterase-3B (PDE-3B). The phosphodiesterase catalyses the breakdown of cAMP to its inactive form, thereby decreasing cAMP levels, which in turn reduces PKA activation and, therefore, also translates into preventing HSL stimulation. Insulin may also suppress lipolysis through phosphorylation of the regulatory subunit of protein phosphatase-1 (PP-1), which once activated rapidly dephosphorylates and deactivates HSL, thus decreasing the lipolytic rate⁽ Reference Jaworski, Sarkadi-Nagy and Duncan ^{63 )}. The anti-lipolytic effect of insulin is observed already minutes upon binding of the hormone to its receptors.

Hormone-mediated control: growth hormone

While insulin repesents the primary anabolic hormone exerting the main influence periprandially, GH operates directly and through stimulation of insulin growth factor-1, insulin and NEFA during stress and fasting⁽ Reference Møller and Jørgensen ^{64 )}. Thus, GH represents a less potent though critically important regulator of lipolysis, which influences body composition, stimulating muscle mass accretion at the same time as reducing adiposity by a direct lipolytic effect using cAMP- and PKA-dependent pathways. GH-deficient individuals can experience up to a 40 % reduction in plasma NEFA and lipolysis that are returned to normal values by GH replacement therapy. Interestingly, GH activates adenylyl cyclase by selectively shifting the G_iα₂ subunit and removing cAMP production inhibition⁽ Reference Yip and Goodman ^{65 )}. Exogenous GH administration produces an increase in NEFA after 2–3 h, thus reflecting a delayed lipolytic effect when compared with that of catecholamines. In this context, small physiological GH pulses reportedly increase interstitial glycerol levels in abdominal and femoral fat⁽ Reference Gravholt, Schmitz and Simonsen ^{66 )}. In addition, suppression of the normal nocturnal rise in GH is followed by a reduction in subsequent lipolysis in subcutaneous adipose tissue⁽ Reference Samra, Clark and Humphreys ^{67 )}. Endogenous GH has been shown to play a limited metabolic role during the daily fed–fast cycle, whereas it is essential for the increased lipolytic rate observed with more prolonged fasting⁽ Reference Sakharova, Horowitz and Surya ^{68 )}. Recently, adipocyte-specific disruption of JAK2 (JAK2A) in mice has been shown to result in GH resistance in adipocytes, with reduced lipolysis and increased body fat, thereby offering complementary mechanistic insights into the well-recognised effects of GH on lipid flux⁽ Reference Nordstrom, Tran and Sos ^{69 )}.

Hormone-mediated control: other hormones

Cortisol also exerts a lipolytic effect, which is less potent than that of catecholamines at the same time as being delayed (minutes in the case of adrenaline v. hours for cortisol)⁽ Reference Lafontan ^{62 ,} Reference Djurhuus, Gravholt and Nielsen ^{70 )}. Importantly, the in vivo lipolysis stimulation is counteracted by the corticoid-induced insulin release⁽ Reference Samra, Clark and Humphreys ^{71 ,} Reference Ottosson, Lonnroth and Björntorp ^{72 )}, whereby the net outcome of a short-term treatment with a standard dose of corticosteroids is an increase in abdominal adipose tissue lipolysis, without changes in GH concentrations, hyperglucagonaemia and insulin resistance. While a stimulation of lipolysis in human adipose tissue has been also ascribed to PTH⁽ Reference Bousquet-Melou, Galitzky and Lafontan ^{20 ,} Reference Taniguchi, Kataoka and Kono ^{73 )}, it has also been suggested that a PTH excess may promote weight gain by impeding catecholamine-induced lipolysis⁽ Reference McCarty and Thomas ^{34 )}. Whereas in rodents testosterone up-regulates catecholamine-induced lipolysis⁽ Reference Xu, De Pergola and Bjorntorp ^{74 )}, in humans testosterone in physiological concentrations causes a depot-specific reduction of catecholamine-stimulated lipolysis in subcutaneous fat cells, probably due to reduced protein expression of β₂-adrenoceptors and HSL⁽ Reference Dicker, Ryden and Naslund ^{75 –} Reference Zang, Ryden and Wahlen ^{77 )}. The relevance of androgen signalling in lipolysis regulation became evident from the observation that late-onset obesity development in androgen receptor-null male mice was caused in part by a decreased lipolytic activity⁽ Reference Fan, Yanase and Nomura ^{78 )}. The direct molecular mechanism accounting for the hypertrophic adipocytes and expanded white adipose tissue of these mice depends on an altered lipid homeostasis characterised by a decreased lipolysis but not an increased lipogenesis. Interestingly, transcripts for HSL were strikingly decreased, whereas those for lipogenic genes were unchanged or decreased. Androgens slightly decrease lipoprotein lipase (LPL) activity in human adipose tissue organ cultures, but markedly inhibit adipogenesis in primary preadipocyte cultures obtained from subcutaneous and omental depots of both sexes⁽ Reference Blouin, Nadeau and Perreault ^{79 )}. Thus, the androgenic effects on adipose tissue in men as opposed to women may differ more in terms of the magnitude of their negative impact on adipogenesis and lipid synthesis rather than in the direction of the lipolytic action.

Although commonly acting in rodent fat cells as lipolytic agents via stimulatory GTP-binding protein (G_s protein)-coupled receptors, thyrotropin-stimulating hormone, adrenocorticotropic hormone and α-melanocyte-stimulating hormone are either ineffective or very weak stimulators of lipolysis in human adipocytes⁽ Reference Lafontan ^{62 )}. Neither glucagon nor glucagon-like peptide-1 (GLP-1) has been clearly shown to stimulate lipolysis in vitro. Likewise, no significant effects of glucagon or GLP-1 on lipolytic rate or adipose tissue blood flow following local or experimental intravenous normo- and hyperglucagonaemia have been observed⁽ Reference Bertin, Arner and Bolinder ^{80 ,} Reference Gravholt, Moller and Jensen ^{81 )}. However, during the present decade the role of the GLP-1/GLP-1 receptor system in lipolysis has experienced renewed interest⁽ Reference Sancho, Trigo and Martin-Duce ^{82 )}. A dose-dependent lipolytic effect of GLP-1 in 3T3-L1 adipocytes in a receptor-dependent manner involving downstream adenylate cyclase/cAMP signalling has been shown⁽ Reference Vendrell, El Bekay and Peral ^{83 )}.

Cytokine regulation of lipolysis

Cytokine release by both adipocytes and stromovascular cells underlies the participation of adipose tissue in a dynamic cross-talk and potent feedback signalling with key neuroendocrine organs involved in the regulation of food intake, lipid metabolism, glucose disposal, energy expenditure and the stress response⁽ Reference Gannagé-Yared, Yaghi and Habre ^{111 ,} Reference Trayhurn, Drevon and Eckel ^{112 )}. The complex secretory activities of adipose tissue also contribute to the development of insulin resistance and atherogenic processes⁽ Reference Berg and Scherer ^{113 –} Reference Arner, Bernard and Salehpour ^{115 )}. The release of cytokines further exerts important local autocrine and paracrine effects, mainly involved in adipose tissue remodelling, adipogenesis, angiogenesis, inflammation and immunity. Noteworthy, cytokines, like TNF-α, as well as some interleukins and adipokines, are important regulators of spontaneous lipolysis.

Cytokine regulation of lipolysis: TNF-α

TNF-α is produced in large amounts by adipocytes and other cell types within adipose tissue⁽ Reference Frühbeck, Gómez-Ambrosi and Muruzábal ^{84 ,} Reference Arner, Rydén and Arner ^{116 )}. In humans, contrarily to rodents, TNF-α is not released from adipose tissue into the circulation but rather acts predominantly as a local factor⁽ Reference Langin and Arner ^{117 –} Reference Cawthorn and Sethi ^{119 )}. As with other lipolytic agents, important species differences have also been observed as regards TNF-α action. TNF-α is able to stimulate lipolysis by at least three separate mechanisms⁽ Reference Langin and Arner ^{117 ,} Reference Ryden and Arner ^{120 ,} Reference González-Yanes and Sánchez-Margalet ^{121 )}. First, it inhibits insulin receptor signalling, thereby counteracting the anti-lipolytic effect of the hormone. In this respect, TNF-α operates via the inactivation of IRS-1. This can be brought about by the inhibition of tyrosine phosphorylation and by a reduction in the amount of IRS-1 in adipocytes. In fact, TNF-α counteracts tyrosine phosphorylation by promoting serine phosphorylation of IRS-1. The most important TNF-α effect on adipocyte IRS-1 is mediated through the p42/44 mitogen-activated protein (MAP) kinase (Fig. 2). Second, TNF-α is able to stimulate lipolysis by inhibiting the G_i-protein-coupled adenosine receptor signalling to counteract the anti-lipolytic effect of adenosine. TNF-α markedly decreases the protein content of all three G_iα subtypes in rodent fat cells, without changing the amount of G_s protein or β-subunit of the G-protein complex. This decrease in G_i protein mitigates the anti-lipolytic effect of adenosine. Interestingly, TNF-α decreases G_i-protein content through an induction of protein degradation by the proteasomal pathway⁽ Reference Botion, Brasier and Tian ^{122 )}. However, the TNF-α–G_i interaction appears to be specific for rodents because it has not been observed in human fat cells. The third way by which TNF-α induces lipolysis is via direct stimulation of basal lipolysis through interactions with the lipid-binding protein perilipin. Only TNF-α receptor 1 and MAP kinases promote lipolytic effects in fat cells leading to phosphorylation and decreased production of perilipin, the adipose lipid droplet coating protein that protects it from being hydrolysed by HSL⁽ Reference Langin and Arner ^{117 ,} Reference Xu and Hotamisligil ^{123 ,} Reference Xu, Hirosumi and Uysal ^{124 )}. Three MAP kinases, namely p44/42, Jun kinase (JNK) and p38, are activated by TNF-α in fat cells but only the first two have been linked to lipolysis so far.

Mechanistically, TNF-α can stimulate lipolysis in the absence of insulin, thus providing evidence that it does not simply antagonise the anti-lipolytic effects of insulin. Moreover, extracellular glucose is required for the TNF-α-induced lipolytic effect, suggesting that a certain nutritional state or substrate availability is required⁽ Reference Cawthorn and Sethi ^{119 )}. The downstream signals of the TNF-α receptor 1-dependent pathway involve the activation of extracellular signal-related kinases (ERK1/2), JNK, AMP-activated protein kinase (AMPK), inhibitor of κB kinase (IKK) and PKA⁽ Reference Cawthorn and Sethi ^{119 ,} Reference Zhang, Halbleib and Ahmad ^{125 ,} Reference Souza, Palmer and Kang ^{126 )}. However, in fat cells the TNF-α-induced activation of ERK1/2, JNK and IKK is rapid and transient, while TNF-α-induced lipolysis takes more than 6 h, suggesting the existence of more distant events that are likely to be controlled by transcriptional regulation⁽ Reference Cawthorn and Sethi ^{119 ,} Reference Jager, Gremeaux and Gonzalez ^{127 )}.

Cytokine regulation of lipolysis: IL-6 and IL-15

The IL-6 receptor and glycoprotein 130, key elements of the cytokine pathway, are expressed in human adipocytes, pointing to a direct autocrine/paracrine action of IL-6 on fat cells⁽ Reference Lafontan ^{62 )}. Infusions of recombinant human IL-6 have been reported to increase plasma NEFA and glycerol concentrations, leading the authors to conclude that IL-6 represents a novel lipolytic factor that operates as a potent stimulator of lipolysis⁽ Reference van Hall, Steensberg and Sacchetti ^{128 ,} Reference Yang, Ju and Zhang ^{129 )}. Interestingly, IL-6 infusions were accompanied by parallel increases in plasma cortisol and adrenaline levels, whereas the potential effect on GH concentrations was not analysed. In this regard, it is difficult to establish whether the increased lipolysis depends on the direct action of IL-6 or rather reflects the effects of other lipolytic factors such as GH, cortisol and noradrenaline⁽ Reference Jensen ^{130 )}. A more recent study has shown that higher circulating IL-6 concentrations are associated with an increased isoproterenol-stimulated lipolysis especially in omental adipocytes in women⁽ Reference Morisset, Huot and Legare ^{131 )}. In any case, the reported effect on lipolysis of IL-6 is relatively modest compared with that elicited by catecholamines and insulin. The potential involvement of IL-6 during the practice of exercise or other situations related to severe illness, where a clear need for an elevated lipid fuel takes place, has been set forward⁽ Reference Holmes, Watt and Febbraio ^{132 ,} Reference Hiscock, Fischer and Sacchetti ^{133 )}.

Another member of the interleukin family has been proposed to participate in the modulation of lipolysis. The administration of IL-15 has been shown to produce a significant reduction in white adipose tissue via both a decreased rate of lipogenesis and a reduction in LPL activity, without a concomitant decrease in food intake⁽ Reference Carbo, Lopez-Soriano and Costelli ^{134 )}. Comparative studies with other cytokines revealed that IL-15 is apparently more potent in its acute stimulation of lipolysis than IL-6 and TNF-α⁽ Reference Ajuwon and Spurlock ^{135 )}. Noteworthy, when specific inhibitors of PKA or Janus kinase were present an attenuation of the lipolytic effect of IL-15 was observed. IL-15 is known to be highly expressed in skeletal muscle, exerting a potent anabolic effect on muscle protein accretion while decreasing fat depots in adipose tissue⁽ Reference Quinn, Strait-Bodey and Anderson ^{136 )}. Taking these observations together, it can be speculated that IL-15 may operate as a homeorhectic factor that mobilises and directs energy away from the adipocyte to other cells during the acute phase of the inflammatory response.

Interestingly, IL-1β and TNF-α have been shown to activate MAP3K8, also called Tpl2, which is expressed in adipocytes and is implicated in cytokine-induced lipolysis⁽ Reference Jager, Gremeaux and Gonzalez ^{127 )}. Pharmacological inhibition or silencing of Tpl2 was able to prevent MAP kinase kinas/ERK1/2 activation by these cytokines but not by insulin, thereby providing evidence of its involvement in ERK1/2 activation particularly in response to inflammatory stimuli⁽ Reference Jager, Gremeaux and Gonzalez ^{127 )}.

Cytokine regulation of lipolysis: leptin

More than a decade ago the identification of functional leptin receptors (OB-R) in white adipose tissue suggested the involvement of leptin in the direct peripheral regulation of adipocyte metabolism⁽ Reference Frühbeck, Jebb and Prentice ^{137 –} Reference Frühbeck ^{139 )}. In fact, leptin was shown to directly participate in lipid metabolism control through the inhibition of lipogenesis and the stimulation of lipolysis. Leptin reportedly exerts an autocrine–paracrine lipolytic effect on isolated white adipocytes both in vitro and ex vivo ⁽ Reference Frühbeck, Aguado and Martínez ^{140 –} Reference Frühbeck, Gómez Ambrosi and Salvador ^{143 )}.

Adenosine A₁ receptors have been shown to be markedly expressed in adipocytes and influence fat cell metabolism via the regulation of adenylyl cyclase and, therefore, participate in lipolysis control via the inhibitory guanosine 5′-triphosphate (GTP) binding proteins, G_i ⁽ Reference Honnor, Dhillon and Londos ^{144 ,} Reference Honnor, Dhillon and Londos ^{145 )}. The adenosinergic system increases leptin secretion by directly activating adenosine A₁ in white adipose tissue⁽ Reference Rice, Fain and Rivkees ^{146 )}. In this respect, a defective leptin-induced stimulation of lipolysis that opposes the adenosine-mediated tonic inhibition was identified⁽ Reference Frühbeck, Gómez Ambrosi and Salvador ^{143 )}. Interestingly, the lipolytic effect of leptin is located at the adenylyl cyclase-inhibitory G protein step (Fig. 2), providing an explanation for the defective stimulation of adipocyte adenylate cyclase and the blunted lipolysis observed in leptin-deficient and OB-R-lacking rodents as well as in morbidly obese humans⁽ Reference Greenberg, Taylor and Londos ^{147 –} Reference Martin, Klim and Vannucci ^{149 )}. Moreover, storage of surplus energy in white adipose tissue and the development of diet-induced obesity require the blockade of a latent leptin-stimulated energy sump in white adipocytes⁽ Reference Wang, Orci and Ravazzola ^{150 )}. In this regard, the pleiotropic effects of leptin in other metabolically relevant organs like brown adipose tissue, skeletal muscle, pancreas, liver and heart need to be considered⁽ Reference Sáinz, Rodríguez and Catalán ^{108 ,} Reference Gómez-Ambrosi, Frühbeck and Martínez ^{151 –} Reference Huynh, Neumann and Wang ^{157 )}.

Cytokine regulation of lipolysis: adiponectin

Adiponectin, also known as Acrp30, AdipoQ, apM1 or GBP28, is a hydrophilic 30-kDa protein highly expressed and secreted by adipocytes⁽ Reference Fortuño, Rodríguez and Gómez-Ambrosi ^{88 ,} Reference Ahima and Lazar ^{90 )}. The three-dimensional structure of the C-terminal globular domain of adiponectin shows a high structural homology with TNF-α, another well-known lipolytic cytokine⁽ Reference Shapiro and Scherer ^{158 )}. Interestingly, HSL activity has been shown to be positively correlated to adiponectin expression, with percentage body fat and adiponectin mRNA arising as the only independent predictors of adipose tissue HSL activity explaining 26 % of its variability⁽ Reference Bullo, Salas-Salvado and Garcia-Lorda ^{159 )}. Increased adipose tissue mass has been suggested to explain the association between low adiponectin and reduced NEFA tolerance⁽ Reference Lavoie, Frisch and Brassard ^{160 )}. Adiponectin has been shown to inhibit spontaneous and catecholamine-induced lipolysis in human adipocytes of non-obese subjects through AMPK-dependent mechanisms⁽ Reference Wedellova, Dietrich and Siklova-Vitkova ^{161 )}. In contrast to most adipokines, which are markedly up-regulated in obesity, adipose tissue expression and circulating concentrations of adiponectin are decreased in both overweight and obesity, thereby implying a plausibly decreased impact on overall lipolysis. Adiponectin gene knockout mice and primary adipocytes obtained from these mice exhibit an increased lipolysis⁽ Reference Qiao, Kinney and Schaack ^{162 )}. Moreover, adiponectin was shown to suppress HSL activation without modifying adipocyte TAG lipase (ATGL) and comparative gene identification-58 (CGI-58) expression in adipocytes. In addition, adiponectin reportedly reduced the type 2 regulatory subunit RIIα protein levels of PKA by reducing its protein stability, with ectopic expression of RIIα abolishing the inhibitory effects of adiponectin on lipolysis in adipocytes⁽ Reference Qiao, Kinney and Schaack ^{162 )}. The proportion of secreted high-molecular-weight v. total adiponectin has been shown to be higher in visceral than in subcutaneous adipose tissue explants in non-obese individuals, while no differences were observed in obese individuals⁽ Reference Kovacova, Tencerova and Roussel ^{163 )}. More recently, full-length adiponectin was shown to exert an anti-lipolytic effect in non-obese subcutaneous adipose tissue, while the globular and trimeric isoforms exhibited anti-lipolytic activity in obese subcutaneous and visceral adipose tissue, respectively⁽ Reference Wedellova, Kovacova and Tencerova ^{164 )}.

Other elements involved in lipolysis

Analysis of the involvement of other factors in the control of lipolytic pathways is unravelling a huge number of potential modulators, which vary greatly not only in their biochemical structure but also in their main physiological effect and the signalling cascade activated.

Other elements involved in lipolysis: nitric oxide

NO or related redox species have been described to act as regulators of lipolysis both in rodent and human adipocytes⁽ Reference Gaudiot, Jaubert and Charbonnier ^{165 –} Reference Engeli, Boschmann and Adams ^{170 )}. Inhibition of NO release increased lipolysis independently of local blood flow changes. While chemical NO donors stimulate basal lipolysis, they block the characteristic isoproterenol-induced lipolytic activity via the inhibition of adenylyl cyclase and PKA. Inducible NO synthase has emerged as a negative modulator of lipolysis via an oxidative signalling pathway upstream of cAMP production⁽ Reference Penfornis and Marette ^{169 )}.

A functional relationship between leptin and NO has been established in several physiological processes⁽ Reference Frühbeck ^{139 ,} Reference Frühbeck ^{171 –} Reference Becerril, Rodríguez and Catalán ^{175 )}. Given the co-localisation of both factors in fat cells and their involvement in lipolysis, a potential role of NO in the leptin-induced lipolytic effect seemed plausible. In fact, 1 h after exogenous leptin administration a dose-dependent increase in both serum NO concentrations and basal adipose tissue lipolytic rate was observed⁽ Reference Frühbeck, Gómez Ambrosi and Salvador ^{143 )}. Up to 27 % of the variability taking place in lipolysis was attributable to the changes in NO concentrations. The leptin-induced NO production in white adipocytes was shown to be mediated through PKA and MAP kinase activation⁽ Reference Mehebik, Jaubert and Sabourault ^{176 )}. Inhibition of NO synthesis by N ^ω-nitro-l-arginine methyl ester (l-NAME) pretreatment was followed by a reduction in the leptin-mediated lipolysis stimulation compared with leptin-treated control animals. Contrarily, in adipocytes obtained from rats under acute ganglionic blockade, the leptin-induced lipolytic effect did not show differences with the lipolytic rate achieved by leptin in control rats. The NO donor S-nitroso-N-acetyl-penicillamine (SNAP) was able to exert a significant inhibitory effect on isoproterenol-stimulated lipolysis. Thus, NO has emerged as a potentially relevant autocrine–paracrine physiological signal to fine-tune lipolysis by facilitating leptin-induced lipolysis and, at the same time, being able to inhibit catecholamine-induced lipolysis⁽ Reference Frühbeck and Gómez-Ambrosi ^{173 )}.

Other elements involved in lipolysis: natriuretic peptides

Until recently, human fat cell lipolysis was thought to be mediated essentially by a cAMP-dependent PKA-regulated pathway under the control of catecholamines and insulin. However, Lafontan et al. ⁽ Reference Lafontan, Moro and Berlan ^{177 )} provided evidence that natriuretic peptides also have the ability to potently stimulate lipolysis in human adipocytes to the same degree as a non-selective β-adrenoceptor agonist. This lipolytic effect is mediated mainly by natriuretic peptide receptor type A through a cyclic GMP-dependent PKG (cGK-I) signalling pathway (Fig. 2) that does not involve PDE-3B inhibition or cAMP production and PKA activity⁽ Reference Sengenes, Berlan and De Glisezinski ^{178 –} Reference Moro, Galitzky and Sengenes ^{182 )}. Noteworthy, in vitro studies have shown that HSL can also be phosphorylated by the cyclic GMP-dependent signalling cascade. In fact, cGK-I phosphorylates perilipin and HSL. Increases in plasma atrial natriuretic peptide levels by physiological (exercise) or pharmacological stimuli are followed by an enhanced lipid mobilisation⁽ Reference Moro, Pillard and de Glisezinski ^{183 ,} Reference Moro, Pasarica and Elkind-Hirsch ^{184 )}. In humans atrial natriuretic peptide also reportedly induces postprandial lipid oxidation, energy expenditure, and concomitantly arterial blood pressure⁽ Reference Birkenfeld, Budziarek and Boschmann ^{185 ,} Reference Moro and Lafontan ^{186 )}. Taken together, this pathway that participates in lipid mobilisation and energy homeostasis becomes especially important during chronic treatment with β-adrenoceptor antagonists, which inhibit catecholamine-induced lipolysis but enhance cardiac atrial natriuretic peptide release.

Other elements involved in lipolysis: endocannabinoid system

Our understanding of the participation of the endocannabinoid system in energy homeostasis has progressed enormously over the past years⁽ Reference Horvath ^{187 –} Reference Moreno-Navarrete, Catalán and Whyte ^{189 )}. In particular, the observation of the presence of G protein-coupled cannabinoid receptor (CB) CB1 receptors in adipocytes provided a clue for the involvement of endocannabinoids in the peripheral control of lipid metabolism⁽ Reference Cota, Marsicano and Tschop ^{190 –} Reference Matias, Gonthier and Orlando ^{193 )}. Selective CB1 antagonism was shown to coordinately induce key genes of the fatty acid catabolic pathway, thereby favouring lipolysis and reducing fat storage in adipose tissue⁽ Reference Jbilo, Ravinet-Trillou and Arnone ^{191 )}. Interestingly, the selective antagonism of CB1 receptors reportedly induced β₃-adrenoceptors and GH receptors at the same time as repressing the expression of catechol-O-methyltransferase, an enzyme involved in the degradation of catecholamines. The reduced expression of this methyltransferase along with the induction of the receptors of two well-known hormones with lipolytic effects further supports the molecular basis for the participation of endocannabinoids in the modulation of lipolysis.

Amides of fatty acids with ethanolamine (FAE) are biologically active lipids participating in a variety of physiological effects, including appetite regulation. While the polyunsaturated FAE anandamide (arachidonoylethanolamide) increases food intake by activating G protein-coupled cannabinoid receptors, the monounsaturated FAE oleoylethanolamide (OEA) reduces feeding as well as body-weight gain and stimulates lipolysis by activating the nuclear receptor PPAR-α⁽ Reference Guzman, Lo Verme and Fu ^{194 ,} Reference Martinez de Ubago, Garcia-Oya and Perez-Perez ^{195 )}.

Other elements involved in lipolysis: ghrelin

Beyond its strong orexigenic effect, the gastrointestinal twenty-eight-amino acid octanoylated peptide ghrelin exerts a wide spectrum of actions including the inhibition of isoproterenol-induced lipolysis in rodent adipocytes⁽ Reference Muccioli, Pons and Ghe ^{196 )}. Both ghrelin and des-acyl ghrelin have been shown to antagonise the catecholamine-stimulated lipolysis via a non-type 1A GH secretagogue receptor. Moreover, acylated and unacylated ghrelin have been also shown to attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase γ and PDE-3B⁽ Reference Baragli, Ghe and Arnoletti ^{197 )}. However, ghrelin infusion in human subjects was observed to induce acute insulin resistance and lipolysis independent of GH signalling⁽ Reference Vestergaard, Gormsen and Jessen ^{198 )}. All of the elements of the ghrelin system have been identified in human adipocytes, including receptors and isoforms as well as the ghrelin-O-acyltransferase or GOAT enzyme⁽ Reference Rodríguez, Gómez-Ambrosi and Catalán ^{199 ,} Reference Rodríguez, Gómez-Ambrosi and Catalán ^{200 )}. Interestingly, in differentiating omental adipocytes, incubation with both acylated and desacyl ghrelin increased PPAR-γ and sterol regulatory element-binding protein-1 mRNA levels, as well as fat storage-related proteins, like acetyl-CoA carboxylase, fatty acid synthase, LPL and perilipin⁽ Reference Rodríguez, Gómez-Ambrosi and Catalán ^{199 )}. Consequently, both ghrelin forms stimulate intracytoplasmatic lipid accumulation at the same time as exhibiting an anti-lipolytic effect.

Other elements involved in lipolysis: other miscellaneous agents

The potent anti-lipolytic effect of nicotinic acid together with its specific binding to adipose tissue was firmly established more than half a century ago⁽ Reference Carlson and Oro ^{201 ,} Reference Carlson and Hanngren ^{202 )}. However, the mechanistic basis for this action on lipolysis control has been provided only more recently⁽ Reference Karpe and Frayn ^{203 )}. Activation of the nicotinic acid receptor triggers an inhibitory G-protein signal, which decreases cAMP concentrations in adipocytes, thereby inhibiting lipolysis. Continuous 24 h nicotinic acid infusion in rats reportedly alters gene expression and basal lipolysis in adipose tissue, producing a NEFA rebound and insulin resistance⁽ Reference Oh, Oh and Choi ^{204 )} that are consistent with clinical observations following treatment with this compound.

Other agents originating from either adipocytes or surrounding cells are known to negatively control adenylyl cyclase activity and inhibit lipolysis via their interaction with plasma membrane receptors belonging to the seven-transmembrane domain receptor family. Autacoid agents, as already mentioned including adenosine, prostaglandins and their metabolites, exert a clear anti-lipolytic effect. Whereas adenosine and neuropeptide Y reportedly inhibit lipolysis, for PGE₂ a biphasic effect has been put forward with nanomolar concentrations suppressing lipolysis, but micromolar levels resulting in lipolysis stimulation⁽ Reference Jaworski, Sarkadi-Nagy and Duncan ^{63 )}. On the contrary, PGI₂ showed no effect or exerted also a biphasic effect, whereby nanomolar concentrations stimulated lipolysis, whereas at micromolar levels lipolysis was suppressed.

Cachexia-inducing tumours produce a lipid-mobilising factor (LMF) that causes an immediate glycerol release when incubated with murine adipocytes, with the stimulation of lipolysis by LMF being associated with an elevation in intracellular cAMP concentrations⁽ Reference Tisdale ^{205 –} Reference Cabassi and Tedeschi ^{207 )}. Zn-α₂-glycoprotein (ZAG), a tumour-related LMF of 43 kDa, has been found to be expressed in 3T3-L1 cells as well as in the major fat depots of mice, being up-regulated in rodents with cancer cachexia⁽ Reference Bing, Bao and Jenkins ^{208 )}. Both ZAG expression and protein have been also detected in human adipocytes of visceral and subcutaneous origin. Remodelling of adipose tissue together with decreased lipid storage constitute a hallmark of cancer patients with cachexia. In addition to ATGL- and HSL-enhanced lipolysis, in cancer other factors such as ZAG have been shown to participate in TAG degradation leading to white adipose tissue atrophy. ZAG expression and release by adipose tissue are up-regulated in weight-losing cancer patients, suggesting that ZAG operates both locally and systemically to stimulate lipid mobilisation⁽ Reference Bing, Mracek and Gao ^{206 )}. However, ZAG did not display the thermogenic effects of the β-adrenoceptor agonist, nor did it increase β₃-adrenoceptor or UCP1 (uncoupling protein 1) gene expression in brown adipose tissue, thereby implying that it does not behave as a typical β_3/2-adrenoceptor agonist⁽ Reference Wargent, O'Dowd and Zaibi ^{209 )}. Thus, ZAG has emerged as a novel adipokine, being identified as an additional adipose tissue factor closely related to body weight loss not only via modulation of lipolysis in fat cells but also by activating AMPK in skeletal muscle cells⁽ Reference Bing, Bao and Jenkins ^{208 ,} Reference Eckardt, Schober and Platzbecker ^{210 )}.

The octapeptide angiotensin II (Ang II) is the active component of the renin–angiotensin system (RAS). A local RAS is present in adipose tissue, with all the elements of the system, including angiotensinogen, renin and angiotensin-converting enzyme, having been identified in adipocytes⁽ Reference Sarzani, Salvi and Dessi-Fulgheri ^{211 )}. Noteworthy, Ang II has been shown to decrease local blood flow in a dose-dependent manner and to inhibit lipolysis in adipose tissue with the effects being similar in both normal-weight and obese individuals⁽ Reference Goossens, Blaak and Saris ^{212 )}. In the last decade evidence has been provided that adipose tissue is a source of vasoactive peptides that further exert metabolic actions⁽ Reference Frühbeck ^{213 )}. Thus, endothelin-1 is a powerful vasoconstrictor primarily produced and secreted by endothelial cells to operate on the underlying vascular smooth muscle cell layer that can also act on adipocytes inducing lipolysis via the ERK pathway⁽ Reference Juan, Chang and Lai ^{214 ,} Reference Juan, Chang and Huang ^{215 )}. In human subjects endothelin-1 has been shown to selectively counteract insulin inhibition of visceral adipocyte lipolysis, decreasing the expression of insulin receptor, IRS-1 and PDE-3B and increasing the expression of the endothelin receptor-B (ET_BR) in visceral but not subcutaneous adipocytes⁽ Reference van Harmelen, Eriksson and Astrom ^{216 )}. The ET_BR-mediated effects were signalled via the PKC and calmodulin pathways. Subsequently, it was further observed that long-term incubation of human adipocytes with endothelin-1 increases lipolysis via the activation of ET_AR⁽ Reference Eriksson, van Harmelen and Stenson ^{217 )}. Likewise, the fifty-two-amino acid vasoactive peptide adrenomedullin together with its receptor components (calcitonin receptor-like receptor and receptor activity modifying protein-2 (CRLR/RAMP2)) have been identified to be concomitantly expressed in adipose tissue (Fig. 2), exhibiting a tissue-specific up-regulation during the development of obesity⁽ Reference Shichiri, Fukai and Ozawa ^{218 ,} Reference Fukai, Yoshimoto and Sugiyama ^{219 )}. Interestingly, in adipose tissue adrenomedullin acts as an autocrine–paracrine factor to regulate lipid mobilisation, inhibiting lipolysis through NO-mediated β-adrenergic agonist oxidation⁽ Reference Harmancey, Senard and Pathak ^{220 )}. In this context, it has been proposed that adrenomedullin alone is devoid of lipolytic function and inhibits β-adrenergic-stimulated lipolysis by shifting the concentration–response curve for isoproterenol by a NO-dependent mechanism; specifically, adrenomedullin-induced NO modifies isoproterenol through an extracellular oxidative reaction to yield its aminochrome, isoprenochrome. However, other studies have provided evidence for adrenomedullin dose-dependently elevating cAMP levels and the lipolytic rate⁽ Reference Iemura-Inaba, Nishikimi and Akimoto ^{221 )}. In this case, adrenomedullin was shown to increase the phosphorylation of PKA, ERK and Akt and would reportedly exhibit additive effects on isoproterenol-induced lipolysis.

Apelin represents a further peptide with vasoactive characteristics that has been subsequently shown to be secreted by adipocytes of both humans and rodents, being up-regulated in states of obesity⁽ Reference Boucher, Masri and Daviaud ^{222 )}. The identification in adipocytes of apelin and the apelin receptor (APJ), a G-protein-coupled receptor, supported a plausible autocrine participation of this peptide in adipobiology. In this line, apelin was shown to dose-dependently stimulate AMPK phosphorylation in human adipose tissue, which was associated with increased glucose uptake⁽ Reference Attane, Daviaud and Dray ^{223 )}. Apelin reportedly decreased isoproterenol-induced NEFA and glycerol release in 3T3-L1 cells and isolated adipocytes abrogating the catecholamine-induced HSL phosphorylation via G-protein q polypeptide (G_q), G_i pathways and AMPK activation⁽ Reference Yue, Jin and Xu ^{224 )}. The apelin-induced inhibition of basal lipolysis was exerted through AMPK-dependent enhancement of perilipin expression by preventing lipid droplet fragmentation and hormone-stimulated acute lipolysis inhibition mediated by decreasing perilipin phosphorylation⁽ Reference Than, Cheng and Foh ^{225 )}. Moreover, apelin also suppressed adipogenesis through MAP kinase kinase/ERK signalling.

Pigment epithelium-derived factor (PEDF) is a 50-kDa protein of the non-inhibitory serpin family of serine protease inhibitors originally identified as a regulator of hepatic TAG metabolism involved in the development of insulin resistance in obesity⁽ Reference Zechner, Zimmermann and Eichmann ^{6 ,} Reference Crowe, Wu and Economou ^{226 ,} Reference Sabater, Moreno-Navarrete and Ortega ^{227 )}. Subsequently it was tested whether this adipocyte-secreted factor also exhibits autocrine–paracrine lipolytic effects. PEDF was shown to stimulate TAG hydrolysis in adipose tissue, muscle and liver via ATGL⁽ Reference Borg, Andrews and Duh ^{228 )}. The exact mechanisms underlying the participation of PEDF in insulin resistance, obesity and non-alcoholic fatty liver disease still need to be fully elucidated⁽ Reference Yamagishi, Amano and Inagaki ^{229 –} Reference Moreno-Navarrete, Touskova and Sabater ^{231 )}. The potential role of other recently identified adipose-related factors on lipolysis such as serum amyloid A, osteopontin, osteocalcin, osteoprotegerin, obestatin, lipocalin 2, visfatin, nerve growth factor-inducible derived peptides, omentin, mammalian chitinase-like protein YKL40, chemerin, vitamin D and tenascin C, among others, beyond their originally reported effects merits to be specifically investigated⁽ Reference Gannagé-Yared, Yaghi and Habre ^{111 ,} Reference Sabater, Moreno-Navarrete and Ortega ^{227 ,} Reference Gómez-Ambrosi, Salvador and Rotellar ^{232 –} Reference Fernández-Real, Izquierdo and Ortega ^{245 )}.

TAG hydrolysis

Only a decade ago the initiation of TAG hydrolysis was thought to be exclusively controlled by HSL⁽ Reference Arner ^{2 –} Reference Young and Zechner ^{7 ,} Reference Shi and Burn ^{253 –} Reference Bezaire and Langin ^{255 )}. However, the generation of Hsl-knockout mice revealed the existence of residual HSL-independent TAG lipase activity, pointing to the existence of previously unidentified adipose tissue lipases. Currently, ATGL is well recognised to be the lipase responsible for initiating TAG breakdown to yield DAG⁽ Reference Girousse and Langin ^{5 ,} Reference Zechner, Zimmermann and Eichmann ^{6 )}. ATGL is a 54-kDa TAG hydrolase, also named phospholipase A2ξ or desnutrin, belonging to the family of patatin-like phospholipase domain-containing proteins (PNPLA) with specificity for TAG as a substrate⁽ Reference Zechner, Zimmermann and Eichmann ^{6 ,} Reference Haemmerle, Lass and Zimmermann ^{248 ,} Reference Zimmermann, Strauss and Haemmerle ^{256 ,} Reference Zechner, Kienesberger and Haemmerle ^{257 )}. Atgl-knockout mice and knockdown studies in adipocytes provided evidence for the involvement of ATGL in TAG but not DAG hydrolysis. Atgl-null mice exhibited a blunted lipolysis, producing a more than 75 % reduction in NEFA release and a significant TAG accumulation in adipocytes leading to obesity⁽ Reference Haemmerle, Lass and Zimmermann ^{248 ,} Reference Schweiger, Schreiber and Haemmerle ^{258 )}. The co-activator of ATGL, CGI-58, also known as α/β-hydrolase domain-containing protein 5 (ABHD5), was shown to stimulate TAG hydrolase activity in wild-type and Hsl-deficient but not Atgl-deficient mice. ATGL and HSL are responsible for 95 % of TAG lipase activity, thereby suggesting a complementary relationship between the two lipases⁽ Reference Zechner, Kienesberger and Haemmerle ^{257 –} Reference Thompson, Lobo and Bernlohr ^{259 )}.

ATGL is highly expressed in adipose tissue, with its expression being profoundly elevated during adipocyte differentiation. Two phosphorylation sites (Ser404 and Ser428) have been identified within the C-terminal region of ATGL. Furthermore, the enzymic activity and its interaction with CGI-58 are dependent on the C-terminal region⁽ Reference Schweiger, Schoiswohl and Lass ^{260 )}. Overexpression of Atgl elevates TAG hydrolysis as well as basal and catecholamine-stimulated lipolysis, while Atgl silencing decreases TAG hydrolase activity, TAG storage and lipid droplet size⁽ Reference Zechner, Kienesberger and Haemmerle ^{257 )}. Alterations of Atgl expression resulted in dramatic changes in whole-cell lipolysis. Conversely, silencing of Atgl or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Taken together, these findings suggest that in humans the ATGL–CGI-58 complex acts independently of HSL and precedes its action in the sequential hydrolysis of TAG.

Fasting, glucocorticoids and PPAR agonists increase Atgl mRNA expression, whereas food intake and insulin decrease it⁽ Reference Lass, Zimmermann and Oberer ^{261 ,} Reference Chakrabarti, Kim and Singh ^{262 )}. Cellular TAG lipolysis by ATGL produces essential mediators involved in lipid ligand generation for PPAR activation, with Atgl deficiency in mice reducing mRNA levels of PPAR-α and PPAR-δ target genes⁽ Reference Haemmerle, Moustafa and Woelkart ^{263 )}. While mammalian target of rapamycin (mTOR)-dependent signalling has been observed to decrease Atgl mRNA expression, FoxO1 activation by SIRT1-mediated deacetylation elevated it⁽ Reference Chakrabarti, Kim and Singh ^{262 ,} Reference Chakrabarti and Kandror ^{264 –} Reference Chakrabarti, English and Karki ^{266 )}. However, the role of AMPK in lipolysis control remains controversial⁽ Reference Yin, Mu and Birnbaum ^{267 –} Reference Gaidhu, Bikopoulos and Ceddia ^{271 )}. In this sense, the precise mechanisms of ATGL regulation need to be fully established. Recently, a protein encoded by the G0/G1 switch gene 2 (G0S2) has been identified as a selective regulator of ATGL by attenuating its action both in vitro and in vivo ⁽ Reference Yang, Lu and Lombès ^{272 ,} Reference Schweiger, Paar and Eder ^{273 )}. G0S2 is highly expressed in adipose tissue and differentiated adipocytes interacting specifically with ATGL to inhibit its TAG hydrolase activity. While knockdown of endogenous G0S2 enhances both basal and stimulated lipolysis in adipocytes, overexpression of G0S2 decreases the lipolytic rate of adipocytes and adipose tissue explants. G0S2 has been further shown to regulate human lipolysis influencing ATGL activity and intracellular localisation by anchoring the lipase to lipid droplets (Fig. 3) independently of the C-terminal lipid-binding domain of ATGL⁽ Reference Schweiger, Paar and Eder ^{273 )}. Moreover, G0S2 expression has been observed to be diminished in poorly controlled type 2 diabetes, thereby establishing a potential link between adipose tissue G0S2 down-regulation and insulin resistance. Given that the above-mentioned characteristics reveal ATGL as an attractive therapeutic target, the development and characterisation of a selective small-molecule inhibitor of ATGL, atglistatin, may prove of interest for the pharmacological treatment of dyslipidaemic and metabolic disorders⁽ Reference Mayer, Schweiger and Romauch ^{274 )}.

Fig. 3 Schematic representation of basal (a) and stimulated (b) lipolysis, the catabolic pathway by which TAG are hydrolysed into fatty acids (FA). AC, adenylyl cyclase; ATGL, adipocyte TAG lipase; cAMP, cyclic AMP; CGI-58, comparative gene identification-58; DAG, diacylglycerol; FABP4, fatty acid binding protein 4; G0S2, G0/G1 switch gene 2; G_s, stimulatory GTP-binding proteins; HSL, hormone-sensitive lipase; MAG, monoacylglycerol; MGL, monoacylglycerol lipase; P, phosphate; PKA, protein kinase A. (A colour version of this figure can be found online at http://www.journals.cambridge.org/nrr)

Diacylglycerol hydrolysis

HSL, an 84-kDa cytoplasmic protein with demonstrated activity for a wide range of substrates including TAG, DAG, cholesteryl esters and retinyl esters, was presumed to be the rate-limiting enzyme in the initial steps of the lipolytic process. However, several important findings challenged this view of the unique regulatory and rate-limiting role of HSL on lipolysis, pointing to the existence of alternative lipases targeting TAG molecules to counterbalance the strong affinity of HSL for DAG⁽ Reference Lafontan and Langin ^{4 ,} Reference Girousse and Langin ^{5 ,} Reference Young and Zechner ^{7 ,} Reference Osuga, Ishibashi and Oka ^{250 ,} Reference Zechner, Kienesberger and Haemmerle ^{257 ,} Reference Okazaki, Osuga and Tamura ^{275 )}: (i) PKA-dependent HSL phosphorylation led only to a 2- to 3-fold increase in TAG hydrolase activity, while whole-cell lipolysis resulted in a 100-fold increase; (ii) Hsl-null mice exhibited a normal body weight with decreased adiposity; (iii) these mutants further showed DAG adipocyte accumulation; (iv) the existence of residual TAG hydrolase activity and lipolysis despite HSL silencing or specific pharmacological inhibition; and (v) failure of HSL overexpression to promote whole-cell lipolysis. As mentioned previously, the identification of ATGL provided explanations for these findings⁽ Reference Osuga, Ishibashi and Oka ^{250 ,} Reference Granneman and Moore ^{254 ,} Reference Haemmerle, Zimmermann and Strauss ^{276 )}.

Fig. 3 illustrates ATGL and HSL regulation in basal and stimulated conditions. ATGL and HSL have the capacity to hydrolyse in vitro the first ester bond of TAG. ATGL exhibits 10-fold higher substrate specificity for TAG than DAG, selectively enabling the first step in TAG hydrolysis, leading to the formation of DAG and fatty acid. An important step in lipolysis activation comprises the translocation of HSL from a cytosolic compartment to the surface of the lipid droplet. Upon lipolytic stimulation, HSL moves from the cytosol to the surface of lipid droplets where it interacts with perilipin-1 and neutral lipids. Noteworthy, adipocytes lacking perilipin-1 are incapable of translocating HSL to the lipid droplet after increases in cAMP⁽ Reference Holm ^{277 ,} Reference Peyot, Nolan and Soni ^{278 )}. Perilipin-1 operates as a dynamic scaffold to coordinate the access of enzymes to the lipid droplet in a way that is responsive to the metabolic state of the adipocyte⁽ Reference Tansey, Sztalryd and Gruia-Gray ^{279 ,} Reference Tansey, Sztalryd and Hlavin ^{280 )}. Thus, in basal conditions (Fig. 3(a)) perilipin-1 limits lipase access to the lipid droplet⁽ Reference Sztalryd, Xu and Dorward ^{281 )}. Lipolysis stimulation is followed by HSL translocation from the cytosol to lipid droplets and redistribution of ATGL, resulting in enriched colocalisation of the two lipases. Interestingly, the ATGL–CGI-58 complex acts independently of HSL and precedes its action in the sequential hydrolysis of TAG in humans. The increased number of ATGL–CGI-58 complexes formed following perilipin-1 phosphorylation (which releases CCI-58) and docked on small lipid droplets govern PKA-stimulated lipolysis (Fig. 3(b)). The association between fatty acid binding protein 4 (FABP4) and HSL represents a further regulatory step. Fatty acid binding to FABP4 and HSL phosphorylation precede the association of FABP4 and HSL. FABP4 also participates in the trafficking of fatty acids from the site of hydrolysis (i.e. the lipid droplet) to the plasma membrane. In addition to supporting fatty acid trafficking to the plasma membrane in a reaction that is independent of its physical association with HSL, FABP4 bound to fatty acids associates with activated, phosphorylated HSL on the surface of lipid droplets. The sequential effect of ATGL-accentuated TAG hydrolysis, phosphorylated HSL and MGL activity yields massive increases in NEFA release in response to PKA activation.

The expression profile of HSL basically mirrors that of ATGL, given that both enzymes coordinatedly hydrolyse TAG and, therefore, share some regulatory characteristics but differ in the mechanisms of enzyme control⁽ Reference Zechner, Zimmermann and Eichmann ^{6 )}. Whereas β-adrenergic stimulation exerts ATGL regulation mainly via CGI-58 recruitment, HSL constitutes the main target for PKA-catalysed phosphorylation⁽ Reference Belfrage, Fredrikson and Olsson ^{282 )}. Adipocyte HSL encompasses an N-terminal domain (that interacts with FABP4) and a C-terminal catalytic domain (that contains the active site as well as a regulatory module with all the known phosphorylation sites of HSL)⁽ Reference Lafontan and Langin ^{4 ,} Reference Bezaire and Langin ^{255 ,} Reference Ray, Beylot and Arner ^{283 )}. Phosphorylation of HSL at Ser563, Ser659 and Ser660 by PKA and at Ser660 via the ERK pathway activate lipolysis⁽ Reference Greenberg, Shen and Muliro ^{284 )}. The PKA-dependent lipolytic effect is exerted increasing HSL's intrinsic activity and promoting its access to TAG molecules within the adipocyte. Conversely, AMPK exerts an anti-lipolytic effect, blocking the translocation of HSL to the lipid droplets by its phosphorylation at Ser565⁽ Reference Lass, Zimmermann and Oberer ^{261 )}. Deactivation of lipolysis mediated by insulin is associated with down-regulation of HSL and ATGL expression⁽ Reference Kralisch, Klein and Lossner ^{285 ,} Reference Kershaw, Hamm and Verhagen ^{286 )}. Moreover, insulin signalling phosphorylates and activates PDE isoforms via PKB, cAMP hydrolysis and PKA inhibition, resulting in the prevention of HSL and perilipin-1 phosphorylation, HSL activation and translocation as well as CGI-58-mediated ATGL activation. The peripheral control of insulin is accompanied by a central mechanism via the sympathetic nervous system that reduces the activitiy of both HSL and ATGL⁽ Reference Scherer, O'Hare and Diggs-Andrews ^{287 )}.

Monoacylglycerol hydrolysis

The final step of lipolysis is catalysed by MGL, which is constitutively expressed in adipose tissue and has no affinity for DAG, TAG or cholesteryl esters⁽ Reference Bezaire and Langin ^{255 )}. The enzymic activity of MGL is required in the final hydrolysis of the 2-monoacylglycerols produced by HSL activation. Site-directed mutagenesis has shown the relevance of Ser122, Asp239 and His269 in the lipase and esterase activities of MGL⁽ Reference Bezaire and Langin ^{255 ,} Reference Taschler, Radner and Heier ^{288 )}.

Other lipases

The contribution of alternative lipases to ATGL and HSL to the overall lipolytic capacity and maintenance of the highly dynamic TAG turnover has yet to be completely discerned. Potential TAG hydrolases have been identified within members of the carboxylesterase/lipase and the patatin homology domain families⁽ Reference Zechner, Zimmermann and Eichmann ^{6 )}. Carboxylesterase-3/TAG hydrolase-1 is supposedly involved in HSL-independent lipolysis in adipocytes and participates in the assembly and secretion of VLDL in the liver ⁽ Reference Soni, Lehner and Metalnikov ^{289 ,} Reference Wei, Ben Ali and Lyon ^{290 )}. Among the patatin homology domain family, PNPLA4 and PNPLA5 have been observed to exhibit TAG hydrolase, DAG transacylase and retinylester hydrolase activity in vitro, which needs to be confirmed in vivo ⁽ Reference Kienesberger, Oberer and Lass ^{291 )}. Noteworthy, the member with the highest ATGL homology is PNPLA3 or adiponutrin⁽ Reference Polson and Thompson ^{292 –} Reference Basantani, Sitnick and Cai ^{295 )}.

Lipid droplet proteins

Cytoplasmic lipid droplets are organelles in which cells store neutral lipids for use as an energy source in times of need, but they also play important roles in the regulation of key metabolic processes, with excess accumulation of intracellular lipids being associated with obesity, type 2 diabetes and atherosclerosis. Fat droplets may constitute up to 95 % of the total adipocyte volume, being mainly composed by TAG. Intracellular TAG storage droplets have emerged as extraordinarily dynamic organelles, with signalling events underlying lipid mobilisation by shuttling protein trafficking to a specialised subset of these droplets⁽ Reference Greenberg, Coleman and Kraemer ^{15 )}. Thus, lipid droplet scaffold proteins are key elements in organising and directing the lipolytic signalling cascade⁽ Reference Greenberg, Coleman and Kraemer ^{15 ,} Reference Bays, González-Campoy and Bray ^{246 )}.

The function of lipid droplets is regulated by their coating proteins, collectively termed PAT proteins after perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kDa (TIP47)⁽ Reference Lafontan and Langin ^{4 ,} Reference Brasaemle ^{296 ,} Reference Brasaemle, Subramanian and Garcia ^{297 )}. Further members of the family are S3-12, oxidative tissue-enriched PAT protein (OXPAT), myocardial lipid droplet protein (MLDP) and lipid storage droplet protein 5 (LSDP5)⁽ Reference Robenek, Robenek and Buers ^{298 ,} Reference Robenek, Robenek and Troyer ^{299 )}. The members of this family share varying levels of sequence similarity, lipid droplet association and functions in stabilising lipid droplets.

Lipid droplet proteins: perilipin

Lipid droplets in most tissues are coated by two or more members of the perilipin family, which are now numbered according to the order of discovery⁽ Reference Kienesberger, Oberer and Lass ^{291 )}. Expression of perilipin-1 is mainly restricted to white and brown adipocytes and, to a lesser extent, steroidogenic cells of adrenal cortex, testes and ovaries. Perilipin-2 (formerly adipophilin or ADRP) and perilipin-3 (formerly TIP47) are ubiquitously expressed and, therefore, lipid droplet components of most tissues. While perilipin-4 (formerly S3-12) is primarily expressed in white adipocytes, perilipin-5 (formerly OXPAT, MLDP, or LSDP5) is expressed in brown adipocytes as well as myocytes of skeletal muscle and heart, all of which rely on lipolysis to provide fatty acids to mitochondria for β-oxidation to drive either ATP production or heat generation. Thus, the perilipin composition of lipid droplets within a specific tissue constitutes an important component of lipolysis regulation.

Perilipin is the best-known member of the PAT family, with perilipin-1 being the predominant isoform found in mature adipocytes, the most abundant protein on the lipid droplet surface and the major substrate for cAMP-dependent PKA in lipolytically stimulated adipocytes⁽ Reference Brasaemle, Subramanian and Garcia ^{297 ,} Reference Londos, Brasaemle and Schultz ^{300 –} Reference Yang, Heckmann and Zhang ^{308 )}. Perilipin limits the access of cytosolic lipases to lipid droplets, thereby facilitating TAG storage under basal conditions (Fig. 3(a)). When energy is needed, perilipin is phosphorylated by PKA, facilitating maximal lipolysis by ATGL and HSL (Fig. 3(b)). Thus, perilipin expression and its phosphorylation state are key in lipolysis control, with phosphorylation of Ser492 producing a lipid droplet remodelling, widely increasing the surface area for lipase binding, while Ser517 is essential for ATGL-dependent lipolysis in stimulated conditions⁽ Reference Lafontan and Langin ^{4 )}. Perilipin-1 is also phosphorylated by the cyclic GMP-dependent PKG.

Perilipin ablation confers resistance to genetic or diet-induced obesity, producing a lean phenotype with smaller adipocytes, increased basal lipolysis and attenuated stimulated lipolysis⁽ Reference Martínez-Botas, Anderson and Tessier ^{301 )}. Recently, perilipin-1 has been shown to move between the fat droplet and the endoplasmic reticulum⁽ Reference Skinner, Harris and Shew ^{309 )}, which is physiologically reasonable given that lipid droplets are largely derived from the endoplasmic reticulum. In this regard, perilipin-mediated lipid droplet formation in adipocytes was demonstrated to promote sterol regulatory element-binding protein-1 (SREBP-1) processing and TAG accumulation, suggesting an interplay between lipid droplet formation and SREBP-1 activation via a positive feedback loop⁽ Reference Takahashi, Shinoda and Furuya ^{310 )}. Therefore, the lysosomal protein degradation machinery of perilipin may constitute a target mechanism for enhancing adipocyte lipolysis. Interestingly, a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells highlighted the relevance of elements of the vesicle-transport systems in lipolysis regulation through the identification of the vesicle-mediated coat protein complex I (COPI) as an evolutionary-conserved regulator of PAT protein composition at the lipid droplet surface⁽ Reference Beller, Sztalryd and Southall ^{311 ,} Reference Guo, Walther and Rao ^{312 )}. In addition to regulating PAT protein composition, COPI promotes the association of ATGL with the lipid droplet surface to mediate lipolysis. These genes are conserved in mammalian cells, thus suggesting that a similar complex might be operative in adipocytes. Although COPI-mediated transport reportedly participates in delivery of ATGL to the lipid droplet surface, depletion of β-COP (a subunit of the COPI coat complex) does not affect association of ATGL with lipid droplets or ATGL-mediated lipolysis, pointing to the possibility of alternative transport mechanisms implicated in the regulation of lipid homeostasis⁽ Reference Takashima, Saitoh and Hirose ^{313 )}.

Lipid droplet proteins: coactivator comparative gene identification-58 (CGI-58) or α/β-hydrolase domain-containing protein 5 (ABHD5)

CGI-58 lacks lipase activity in itself but potently and selectively stimulates lipolysis by activating ATGL. As mentioned above, in basal unstimulated conditions CGI-58 binds tightly to lipid droplets by interacting with perilipin-1 and is unable to activate ATGL⁽ Reference Lafontan and Langin ^{4 )}. However, following β-adrenoceptor stimulation CGI-58 is quickly dispersed to the cytosol, favouring ATGL co-localisation and migration to small lipid droplets. Thus, under stimulated conditions, the intracellular cAMP elevation and PKA activation promote perilipin-1 phosphorylation, which is followed by the dissociation from perilipin of CGI-58, which subsequently interacts with ATGL and activates TAG hydrolysis (Fig. 3(b)). In addition to ATGL activation, a further physiological function for CGI-58 in phospholipid synthesis with lysophosphatidic acid acyltransferase activity has been observed⁽ Reference Lafontan and Langin ^{4 )}.

Lipid droplet proteins: Cide domain-containing proteins

A further family of lipid droplet-associated proteins encompasses the cell death-inducing DFFA (DNA fragmentation factor-α)-like effectors (Cide), which includes three members (Cidea, Cideb and Cidec/Fsp27) with tissue-specific expression⁽ Reference Girousse and Langin ^{5 )}. In spite of Cidea and Cideb not being expressed in white adipose tissue, their deletion yielded rodents with lower body weight and improved insulin sensitivity as well as resistant to diet-induced obesity⁽ Reference Zhou, Yon Toh and Chen ^{314 ,} Reference Li, Ye and Xue ^{315 )}. In the Cidea knockout model the elevated energy expenditure was attributable to brown adipose tissue via enhanced AMPK activity leading to increased fatty acid oxidation⁽ Reference Qi, Gong and Zhao ^{316 )}. The Cideb mutants exhibited a decreased hepatic VLDL secretion and de novo fatty acid oxidation related to enhanced hepatic oxidative activity⁽ Reference Ye, Li and Liu ^{317 ,} Reference Tiwari, Siddiqi and Siddiqi ^{318 )}. Cidea is also involved in human adipocyte lipolysis, TAG deposition and fatty acid oxidation via cross-talk with TNF-α, which inhibits the transcription of the gene⁽ Reference Nordstrom, Ryden and Backlund ^{319 –} Reference Christianson, Boutet and Puri ^{321 )}. Cidea co-localises with perilipin around lipid droplets in fat cells. An increased lipolysis is observed in Cidea-depleted human adipocytes. Contrarily, ectopical expression of Cidea in preadipocytes markedly enhances lipid droplet size, promoting lipid accumulation⁽ Reference Puri, Ranjit and Konda ^{322 )}. Noteworthy, Cidea expression is elevated in human cancer cachexia, exhibiting a correlation with elevated NEFA concentrations and weight loss⁽ Reference Laurencikiene, Stenson and Arvidsson Nordstrom ^{323 )}. In humans Cidec, also referred to as fat-specific protein 27, FSP27, is predominantly expressed in subcutaneous adipocytes, being down-regulated in response to a reduced energy intake⁽ Reference Magnusson, Gummesson and Glad ^{324 )}. Small interfering RNA-mediated knockdown of Cidec translated into an increased basal release of NEFA, and decreased responsiveness to adrenergic lipolysis stimulation⁽ Reference Lafontan and Langin ^{4 ,} Reference Ranjit, Boutet and Gandhi ^{325 )}. The interaction between the diverse lipases is also starting to be unfolded. FSP27 and perilipin-1 interaction promotes the formation of large lipid droplets in human adipocytes⁽ Reference Kim, Cho and Yun ^{326 –} Reference Nian, Sun and Yu ^{329 )}. Recently, the unilocular to multilocular transformation that takes place during ‘browning’ of white adipose tissue has been related to Cide-triggered dynamic changes in lipid droplet-associated proteins⁽ Reference Barneda, Frontini and Cinti ^{330 )}.

Lipid droplet proteins: other proteins (GPIHBP1 and Rab)

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) is a 28-kDa glycosylphosphatidylinositol-anchored glycoprotein located on the luminal surface of endothelial cells in tissues where lipolysis takes place such as adipose tissue, skeletal muscle and heart⁽ Reference Young and Zechner ^{7 ,} Reference Muller, Wied and Walz ^{331 )}. The expression of GPIHBP1 in mice is modulated by fasting and refeeding as well as by PPAR-γ agonists. GPIHBP1 knockout mice exhibit chylomicronaemia, even on a low-fat diet, with highly elevated plasma TAG concentrations⁽ Reference Muller, Jung and Wied ^{332 –} Reference Adeyo, Goulbourne and Bensadoun ^{334 )}. GPIHBP1 is highly expressed in the same tissues that express high levels of LPL, namely, heart, adipose tissue, and skeletal muscle where it binds both LPL and chylomicrons, suggesting that GPIHBP1 functions as a platform for LPL-dependent lipolytic processing of TAG-rich lipoproteins, stabilising LPL without activating it.

Rab GTPases, which are key regulators of membrane trafficking, have emerged as particularly relevant molecules in the highly dynamic cellular interactions involved in lipid mobilisation. In this sense, proteomic analyses have consistently identified the small GTPase Rab18 as a component of the lipid droplet coat⁽ Reference Peinado, Pardo and de la Rosa ^{335 )}. Thus, Rab18 provides an excellent marker to follow the dynamics of lipid droplets in living cells as well as to gain insight into the complex regulatory mechanisms involved in lipid storage and release⁽ Reference Malagon, Cruz and Vazquez-Martinez ^{336 –} Reference Pulido, Rabanal-Ruiz and Almabouada ^{338 )}. In 3T3-L1 adipocytes, stimulation of lipolysis increases the association of Rab18 with lipid droplets, suggesting that Rab18 recruitment is regulated by the metabolic state of individual lipid droplets. Furthermore, Rab1a and its effector protein are reportedly involved in the CD36 trafficking signalling pathway⁽ Reference Thompson, Lobo and Bernlohr ^{259 )}.

Integral membrane proteins and transporters

While the main signalling cascades and regulators of lipolysis have been identified, the cellular interactions involved in lipid mobilisation and release still remain to be completely disentangled. Except in adipocytes, lipid droplets are normally small, mobile and interact with other cellular compartments in cells. On the contrary, fat cells are composed mainly of very large, immotile lipid droplets. The striking morphological differences between lipid droplets in adipocytes and non-adipocytes suggest that key differences must exist in the way in which lipid droplets in different cell types interact with other organelles to facilitate lipid transfer. A plethora of molecules involved in these interactions are now emerging, with integral membrane proteins and fatty acid transporters standing out as pivotal elements operating at the dynamic plasma membrane–lipid droplet interface.

Integral membrane proteins and transporters: aquaporin-7

Aquaporins (AQP) are integral membrane proteins that function mainly as water channels. AQP7 belongs to the subfamily of aquaglyceroporins, which are permeable to both glycerol and water, being expressed in adipocytes⁽ Reference Frühbeck ^{339 –} Reference Walker, Holness and Gibbons ^{341 )}. Mouse and human AQP7 exhibit six prospective sites for PKA phosphorylation, suggesting a putative cAMP/PKA-dependent regulation. Aqp7-knockout mice show defective glycerol exit from fat cells, adipocyte hypertrophy due to TAG accumulation and moderate adult-onset obesity⁽ Reference Hara-Chikuma, Sohara and Rai ^{342 ,} Reference Hibuse, Maeda and Nagasawa ^{343 )}. Short-term regulation and translocation of AQP7 to the plasma membrane is stimulated by catecholamines, while insulin exerts a long-term negative control. More recently, in addition to AQP7, the presence and functionality of other members of the aquaglyceroporin subfamily, AQP3 and AQP9, have been identified in adipose tissue and shown to be regulated by insulin and leptin via the PI3K/Akt/mTOR signalling cascade⁽ Reference Rodríguez, Catalán and Gómez-Ambrosi ^{344 )}.

Integral membrane proteins and transporters: caveolin-1

Caveolae account for over 25 % of the adipocyte's membrane, being specialised plasma membrane microdomain invaginations involved in important cellular transport processes such as endo- and transcytosis as well as signal transduction⁽ Reference Frühbeck, López and Diéguez ^{345 )}. Three classes of caveolae formed by caveolin-1, the scaffolding hairpin-like protein facing the cytosol, have been identified, with high-density caveolae taking up exogenous fatty acids and converting them to TAG. These TAG-metabolising caveolae serve as a platform for FABP4, fatty acid transport protein (FATP) 1 and 4 (FATP1 and FATP4), long-chain acyl-CoA synthetase 1 (ACSL1) and CD36 (also known as fatty acid translocase). Noteworthy, these caveolae contain FATP1 and FATP4 together with the enzymes needed for TAG synthesis⁽ Reference Trigatti, Anderson and Gerber ^{346 –} Reference Meshulam, Breen and Liu ^{348 )}. Furthermore, HSL and perilipin have been shown to be associated to these caveolae⁽ Reference Cohen, Razani and Schubert ^{349 )}, demostrating that TAG can be hydrolysed in them (Fig. 4). Caveolin-1 exerts an indirect structural role in caveolae formation, controlling surface availability or stability of CD36, a fatty acid transporter key to long-chain fatty acid uptake⁽ Reference Covey, Brunet and Gandhi ^{350 )}. In response to NEFA, caveolin-1 reportedly translocates from the plasma membrane to lipid droplets. Caveolin-1 knockout mice lack caveolae in adipocyte plasma membranes, exhibiting increased circulating NEFA and TAG, reduced adipocyte lipid droplet size and resistance to diet-induced obesity⁽ Reference Cohen, Razani and Wang ^{351 )}. Experiments with caveolin-1-null mouse embryonic fibroblasts indicate that caveolin-1 deficiency is followed by a total loss of caveolae, absence of CD36 plasma membrane expression and a reduction in fatty acid uptake, which is reverted by re-expression of caveolin-1⁽ Reference Ring, Le Lay and Pohl ^{352 )}. Interestingly, caveolin-1 has been shown to exert inhibitory interactions with various proteins such as PKA, endothelial NOS and insulin receptors, with knockout mice exhibiting an attenuated lipolytic activity and decreased perilipin phosphorylation⁽ Reference Cohen, Razani and Schubert ^{349 )}. Caveolin-1 potently inhibits cAMP-dependent signalling in vivo, with a direct interaction between caveolin-1 and the catalytic subunit of PKA having been demonstrated both in vitro and in vivo.

Fig. 4 Schematic diagram of a caveola present in the adipocyte's membrane and its participation in lipolysis. ACSL1, acyl coenzyme A synthetase 1; cAMP, cyclic AMP; CD36, fatty acid translocase; FA, fatty acid; FABP, fatty acid binding protein; FATP, fatty acid transport protein; HSL, hormone-sensitive lipase; LD, lipid droplet; NOS, NO synthase; PKA, protein kinase A; PP₁, pyrophosphate. (A colour version of this figure can be found online at http://www.journals.cambridge.org/nrr)

Integral membrane proteins and transporters: fatty acid translocase (CD36)

As mentioned above, CD36 localises to caveolae as well as to intracellular vesicles. CD36 is a glycoprotein belonging to the family of class B scavenger receptors predicted to have two transmembrane domains at the N- and C-terminal, a large extracellular domain loop and two short intracellular cytoplasmic tails⁽ Reference Thompson, Lobo and Bernlohr ^{259 )}. CD36 is expressed in organs with high fatty acid metabolism rates, such as adipose tissue, operating as a NEFA scavenger. Insulin activation of the forkhead transcription factor and AMPK stimulation trigger CD36 translocation from intracellular stores to the plasma membrane, thereby enhancing NEFA uptake. CD36 deficiency is associated with increased basal lipolysis and responsiveness to the anti-lipolytic effect of insulin, with Cd36-null mice exhibiting an impaired fatty acid uptake in metabolic tissues (including adipocytes) and increased plasma NEFA and TAG concentrations⁽ Reference Zhou, Samovski and Okunade ^{353 ,} Reference Vroegrijk, van Klinken and van Diepen ^{354 )}. Knockdown of CD36 by RNAi in 3T3-L1 adipocytes resulted in a profound reduction of both basal and insulin-stimulated NEFA uptake. Conversely, overexpression of CD36 led to mice with decreased adiposity and low circulating levels of NEFA, TAG and cholesterol, suggesting that a strict control of these molecules for an effective lipolysis is required.

Integral membrane proteins and transporters: adipose fatty acid binding protein

FABP4, also known as ALBP and aP2, is a cytosolic lipid-binding protein highly expressed in adipocytes involved in fatty acid and retinoic acid intracellular trafficking⁽ Reference Thompson, Lobo and Bernlohr ^{259 )}. It acts as a molecular chaperone, facilitating NEFA uptake and lipolysis, interacting with HSL and shuttling fatty acids out of adipocytes (Fig. 4). Upon PKA activation the HSL–FABP4 complex translocates to lipid droplets. Consistently with this, in Fabp4-knockout mice basal and stimulated lipolysis are attenuated⁽ Reference Kienesberger, Oberer and Lass ^{291 ,} Reference Scheja, Makowski and Uysal ^{355 –} Reference Wei, Zan and Wang ^{357 )}. Interestingly, Fabp4-null mice have been shown to compensate FABP4 deletion by increasing the expression of other FABP, thereby highlighting that lipolysis seems to be linked to total FABP content rather than to a specific FABP type⁽ Reference Lafontan and Langin ^{4 )}.

Integral membrane proteins and transporters: fatty acid transport protein 1

The underlying mechanism for fatty acid uptake by FATP1, an integral membrane protein of about 71 kDa with a hydrophobic domain at the N-terminal that may be membrane-anchored and other membrane-associated domains peripherally associated with the inner leaflet of the membrane, is still unknown. In response to insulin, FATP1 may translocate to structurally disordered non-lipid raft regions of the plasma membrane. Subsequently, FATP1 may extract fatty acid from the inner membrane leaflet and esterify it to CoA, thereby preventing its efflux and driving a NEFA concentration gradient across the membrane⁽ Reference Wu, Ortegon and Tsang ^{358 ,} Reference Richards, Harp and Ory ^{359 )}. Most of the incoming fatty acids are converted into acyl-CoA and preferentially shunted into TAG synthesis (Fig. 4). Noteworthy, the conversion of incoming long-chain fatty acids to TAG takes place on or around the plasma membrane in rat adipocytes, plausibly linking in a mechanistic way fatty acid influx to TAG synthesis⁽ Reference Thompson, Lobo and Bernlohr ^{259 ,} Reference Liu, Gauthier and Sun ^{360 )}. Knockdown and knockout experiments revealed an absolute requirement for FATP1 in insulin-stimulated fatty acid uptake, whereas FATP1 overexpression led to a fatty acid uptake increase.

Integral membrane proteins and transporters: fatty acid transport protein 4

FATP4 presents a 60 % identity to FATP1 and is expressed in adipose tissue, skin, heart, skeletal muscle, liver, as well as in the small intestine, where it was observed to work in intestinal lipid absorption⁽ Reference Thompson, Lobo and Bernlohr ^{259 ,} Reference Lenz, Marx and Chamulitrat ^{361 )}. FATP4 knockdown in 3T3-L1 adipocytes by RNAi did not affect basal and insulin-stimulated fatty acid uptake. FATP4 knockouts exhibit perinatal lethality due to restrictive dermopathy, suggesting a key role in the formation of the epidermal barrier rather than in fatty acid uptake and intestinal lipid absorption.

Integral membrane proteins and transporters: acyl-CoA synthetase long-chain 1

ACSL1, a 78-kDa membrane protein expressed in adipocytes and localised to various subcellular sites including the plasma membrane, lipid droplets, and GLUT4-containing vesicles, co-localises with FATP1⁽ Reference Thompson, Lobo and Bernlohr ^{259 )}. ACSL1 was found to be involved in the reacylation of fatty acids released from the lipid droplets during basal and hormone-induced lipolysis⁽ Reference Richards, Harp and Ory ^{359 )}. Overexpression of ACSL1 in fibroblasts is followed by an increase in NEFA uptake, thereby supporting a co-operative role in fatty acid transport across the adipocyte plasma membrane⁽ Reference Phillips, Goumidi and Bertrais ^{362 )}. However, knockdown of ACSL1 expression by RNAi in 3T3-L1 adipocytes points to a role in fatty acid efflux but not influx.