Hormone-sensitive lipase

In the 1960s HSL was characterized as a lipolytic enzyme sensitive to adrenaline⁽Reference Bjorntorp and Furman^9, Reference Rizack¹⁰⁾. For the following 30 years HSL remained the undisputed regulator of lipolysis. HSL is highly expressed in WAT⁽Reference Holm, Belfrage and Fredrikson¹¹⁾ and displays in vitro hydrolysis activity for TAG, diacylglycerols (DAG), monoacylglycerols⁽Reference Fredrikson, Stralfors and Nilsson¹²⁾, cholesterol and retinyl esters⁽Reference Grober, Lucas and Sorhede-Winzell^13, Reference Strom, Gundersen and Hansson¹⁴⁾. Its relative affinity is ten times greater for DAG than TAG⁽Reference Fredrikson, Stralfors and Nilsson^12, Reference Langin, Lucas and Lafontan¹⁵⁾ and shows a preference for fatty acids in the sn-1 and sn-3 position of TAG molecules⁽Reference Raclot, Langin and Lafontan¹⁶⁾.

The cloning of HSL in the rat and human subjects⁽Reference Holm, Kirchgessner and Svenson^17, Reference Langin, Laurell and Stenson Holst¹⁸⁾ has provided an insight into its gene and protein structure. The carboxy terminal of HSL harbours the active site and regulatory module of the enzyme⁽Reference Osterlund¹⁹⁾. The amino terminal, although less characterized, appears to be required for protein–protein interaction, notably with fatty acid-binding protein (FABP) 4 (detailed later)⁽Reference Shen, Sridhar and Bernlohr²⁰⁾. As alluded to earlier, HSL action is in part regulated by PKA. Three PKA phosphorylation sites have been identified in rat HSL: Ser⁵⁶³; Ser⁶⁵⁹; Ser⁶⁶⁰⁽Reference Anthonsen, Rönnstrand and Wernstedt²¹⁾. The corresponding sites in human HSL are Ser⁵⁵², Ser⁶⁴⁹ and Ser⁶⁵⁰⁽Reference Contreras, Danielsson and Johansson²²⁾. PKA phosphorylation of rat HSL residue Ser⁵⁶³ appears to regulate intrinsic activity⁽Reference Shen, Patel and Natu²³⁾ while residues Ser⁶⁵⁹ and Ser⁶⁶⁰ favour the translocation of a predominantly cytosolic HSL to LD⁽Reference Su, Sztalryd and Contreras^24–Reference Clifford, Londos and Kraemer²⁷⁾. In human HSL PKA phosphorylation of residues Ser⁶⁴⁹ and Ser⁶⁵⁰ has been shown to be the most important in increasing enzymic activity⁽Reference Krintel, Osmark and Larsen²⁸⁾. The pro-lipolytic effect of PKA on HSL is therefore two-pronged: increasing the enzyme's intrinsic activity; promoting its access to TAG molecules in a whole-cell context.

Additional HSL regulatory pathways include the extracellular signal-regulated kinase and AMP-activated PK(AMPK) pathways. HSL is positively regulated by the extracellular signal-regulated kinase pathway via phosphorylation of Ser⁶⁶⁰⁽Reference Greenberg, Shen and Muliro^29, Reference Zhang, Halbleib and Ahmad³⁰⁾ and negatively regulated by AMPK. AMPK, the cellular energy sensor, is activated by increasing AMP:ATP to restore energy levels⁽Reference Kahn, Alquier and Carling³¹⁾. Once activated AMPK phosphorylates HSL on Ser⁵⁶⁵ in human adipocytes⁽Reference Roepstorff, Vistisen and Donsmark^32, Reference Garton and Yeaman³³⁾, resulting in an anti-lipolytic effect⁽Reference Daval, Diot-Dupuy and Bazin³⁴⁾.

Doubt on the lone regulatory role of HSL in lipolysis slowly grew over the years. First, puzzlement revolved around an important mismatch between HSL activity and adipocyte lipolysis in response to PKA activation. PKA-dependent phosphorylation of HSL leads to a two- to three-fold increase in TAG hydrolase activity, while whole-cell lipolysis increases ⩽100-fold. These contrasting observations suggested additional, yet unidentified, regulatory steps in lipolysis. The critical role of the LD structural protein PLIN would later shed light on this issue (described later). Additionally, DAG accumulation in adipose tissue of HSL-null mice⁽Reference Haemmerle, Zimmermann and Hayn³⁵⁾ suggested the presence of an alternative lipase targeting TAG molecules, possibly to complement the strong affinity of HSL for DAG. The identification of adipose TAG lipase (ATGL) in 2004 (see later) supports more recent findings obtained from HSL manipulation; for example, residual TAG hydrolase activity and lipolysis despite HSL silencing⁽Reference Kershaw, Hamm and Verhagen^36–Reference Bezaire, Mairal and Ribet³⁸⁾ or specific pharmacological inhibition⁽Reference Schweiger, Schreiber and Haemmerle^39–Reference Mairal, Langin and Arner⁴¹⁾ and the failure of HSL overexpression to promote whole-cell lipolysis⁽Reference Bezaire, Mairal and Ribet^38, Reference Lucas, Tavernier and Tiraby⁴²⁾.

Adipose TAG lipase

In 2004 three groups independently identified an additional lipase with TAG hydrolase activity⁽Reference Zimmermann, Strauss and Haemmerle^43–Reference Villena, Roy and Sarkadi-Nagy⁴⁵⁾. ATGL (also known as desnutrin or phospholipase A2ξ) belongs to the family of patatin-like phospholipase domain-containing proteins. It is highly expressed in WAT and brown adipose tissue and to a lower extent in testes, skeletal and cardiac muscle⁽Reference Kershaw, Hamm and Verhagen^36, Reference Zimmermann, Strauss and Haemmerle^43, Reference Villena, Roy and Sarkadi-Nagy^45, Reference Lake, Sun and Li⁴⁶⁾. The carboxy-terminal region of ATGL contains a hydrophobic section permitting protein–lipid interactions⁽Reference Schweiger, Schoiswohl and Lass⁴⁷⁾. Accordingly, in mouse models and COS-7 cell lines native or ectopic ATGL is mostly associated with LD⁽Reference Zimmermann, Strauss and Haemmerle^43, Reference Schweiger, Schoiswohl and Lass^47, Reference Kobayashi, Inoguchi and Maeda⁴⁸⁾. Also within the carboxy-terminal region of ATGL are two phosphorylation sites, Ser⁴⁰⁴ and Ser⁴²⁸⁽Reference Bartz, Zehmer and Zhu⁴⁹⁾. The nature of the PK targeting ATGL and the functional role of such sites are unknown. Last, the enzymic activity of ATGL and its interaction with co-activator comparative gene identification 58 (CGI-58) are dependent on the carboxy-terminal region⁽Reference Schweiger, Schoiswohl and Lass⁴⁷⁾.

Studies with ATGL-null mice have revealed the importance of ATGL in energy homeostasis⁽Reference Haemmerle, Lass and Zimmermann⁵⁰⁾. ATGL-null mice display increased WAT mass and ectopic TAG storage in several tissues, including heart tissue, resulting in premature death. A strong body of evidence has further established the central role of ATLG in lipolysis in murine adipocytes or non-adipocyte cell lines. Overexpression of ATGL increases TAG hydrolysis⁽Reference Villena, Roy and Sarkadi-Nagy^45, Reference Lake, Sun and Li⁴⁶⁾ and basal and isoproterenol-stimulated lipolysis⁽Reference Kershaw, Hamm and Verhagen³⁶⁾ while its silencing decreases TAG hydrolase activity⁽Reference Kershaw, Hamm and Verhagen^36, Reference Zimmermann, Strauss and Haemmerle⁴³⁾, TAG storage and LD size⁽Reference Miyoshi, Perfield and Obin³⁷⁾. Unlike HSL, ATGL hydrolysis capacity is mainly targeted towards TAG. In human adipocytes, however, the in vitro TAG hydrolase capacity of ATGL is lower than that of HSL⁽Reference Mairal, Langin and Arner⁴¹⁾. Nonetheless, ATGL plays a crucial role in orchestrating lipolysis in human adipocytes⁽Reference Bezaire, Mairal and Ribet³⁸⁾. Modulation of ATGL with adenoviral transduction and gene silencing dictates basal and PKA-stimulated lipolysis⁽Reference Bezaire, Mairal and Ribet³⁸⁾. The latter study has also demonstrated, in response to PKA-stimulation, translocation of ATGL from the cytosol to LD and consequently its enrichment with HSL. Collectively, these findings suggest that ATGL and HSL act sequentially, despite their common capacity for TAG hydrolysis. HSL remains the lone enzyme capable of DAG hydrolysis, but DAG supply by ATGL controls PKA-stimulated lipolysis in human adipocytes.

Monoacylglycerol lipase

Monoacylglycerol lipase (MGL) was purified from rat adipose tissue in the 1970s⁽Reference Tornqvist and Belfrage⁵¹⁾. This enzyme is expressed in WAT, lung, liver, kidney, testes, brain and heart⁽Reference Karlsson, Contreras and Hellman⁵²⁾. Despite the in vitro capacity of HSL to hydrolyse monoacylglycerols, the presence of MGL in vivo is required for complete lipolysis⁽Reference Fredrikson, Tornqvist and Belfrage⁵³⁾. MGL hydrolyses the 1(3) and 2-ester bonds of monoacylglycerols at equal rates but has no affinity for DAG, TAG or cholesteryl esters. Site-directed mutagenesis has confirmed the importance of Ser¹²², Asp²³⁹ and His²⁶⁹ in the lipase and esterase activities of MGL⁽Reference Karlsson, Contreras and Hellman⁵²⁾. MGL is not thought to be rate limiting in lipolysis because of its abundance⁽Reference Fredrikson, Tornqvist and Belfrage⁵³⁾.

Perilipins

Over the past 15 years it has become clear that LD are not simple aggregates of lipids but rather dynamic and highly-structured organelles, important for cellular homeostasis and the synthesis of lipid signalling molecules. Providing structure to LD is a family of lipid-coating proteins termed PAT. The PAT family in human adipocytes includes perilipin, adipophilin, tail-interacting protein of 47 kDa, S3-12 and oxidative tissues-enriched PAT protein. The proportion of each lipid-coating protein on LD is altered as LD mature⁽Reference Wolins, Quaynor and Skinner^54, Reference Wolins, Skinner and Schoenfish⁵⁵⁾. Perilipin, which was discovered in 1991⁽Reference Greenberg, Egan and Wek⁵⁶⁾, is highly expressed in WAT and brown adipose tissue⁽Reference Greenberg, Egan and Wek^56–Reference Tansey, Sztalryd and Gruia-Gray⁵⁹⁾ and is the most abundant lipid-coating protein on mature LD⁽Reference Wolins, Quaynor and Skinner⁵⁴⁾. Three isoforms arise from alternate splicing of a single mRNA transcript, PLINA being most abundant in WAT LD⁽Reference Greenberg, Egan and Wek^56, Reference Greenberg, Egan and Wek⁵⁷⁾. Ectopic expression of PLINA in 3T3 L1 preadipocytes naturally devoid of PLIN suggests that PLINA forms a physical barrier around LD to reduce lipase access⁽Reference Brasaemle, Rubin and Harten⁶⁰⁾. Three hydrophobic sequences play a major role in anchoring PLINA to LD⁽Reference Subramanian, Garcia and Sekowski⁶¹⁾ yet it is the amino and carboxy terminals that are critical in promoting TAG storage⁽Reference Garcia, Subramanian and Sekowski⁶²⁾.

Investigations with PLIN-null mice clearly illustrate the regulatory role of PLIN in lipolysis. PLIN-ablated mice are lean, have smaller adipocytes and are resistant to diet-induced obesity⁽Reference Martinez-Botas, Anderson and Tessier^58, Reference Tansey, Sztalryd and Gruia-Gray^59, Reference Sztalryd, Xu and Dorward⁶³⁾. In addition, they exhibit elevated basal lipolysis and attenuated stimulated lipolysis. Interestingly, experiments with mouse embryonic fibroblasts from PLIN–/– mice and COS-7 cells lacking PLIN have shown that HSL fails to translocate to LD in response to β-adrenergic stimulation⁽Reference Sztalryd, Xu and Dorward⁶³⁾. Additionally, live culture cell experiments have demonstrated that PKA activation facilitates fluorescence resonance energy transfer between fluorescent probes fused to HSL and PLIN⁽Reference Granneman, Moore and Granneman⁶⁴⁾. Together, these results not only highlight the regulatory role of PLIN as a physical barrier to HSL, but also suggest that PLIN may provide an HSL-docking site on LD.

PLINA has six serine phosphorylation sites targeted by PKA⁽Reference Clifford, Londos and Kraemer^27, Reference Greenberg, Egan and Wek^56, Reference Nishiu, Tanaka and Nakamura^65, Reference Egan, Greenberg and Chang⁶⁶⁾. Of those sites, residue Ser⁵¹⁷ has been demonstrated to be essential to ATGL-dependent lipolysis in stimulated conditions⁽Reference Miyoshi, Perfield and Souza⁶⁷⁾. However, specific phosphorylation of Ser⁴⁹² in murine adipocytes is also of importance as it causes a remodelling of large LD into a myriad of microLD, independently of lipolysis⁽Reference Marcinkiewicz, Gauthier and Garcia⁶⁸⁾. On phosphorylation PLINA remains on the surface of LD but increased LD surface area following fragmentation facilitates lipolysis. Thus, PLINA limits lipase access to LD in the basal state but provides greater access to lipases in stimulated conditions by docking HSL and promoting fragmentation of LD.

Caveolin-1

Caveolae are small invaginations on cell plasma membranes⁽Reference Palade^69, Reference Patel, Murray and Insel⁷⁰⁾. They are common to many cell types but highly expressed in adipocytes⁽Reference Thorn, Stenkula and Karlsson⁷¹⁾. Caveolin is the marker protein for these structures such that ectopic expression of caveolin results in the formation of invaginations on cellular membranes⁽Reference Parton, Hanzal-Bayer and Hancock⁷²⁾. Caveolae have several putative functions, including participation in signal transduction⁽Reference Drab, Verkade and Elger⁷³⁾, membrane trafficking pathways and NEFA binding and transport⁽Reference Trigatti, Anderson and Gerber⁷⁴⁾. Interestingly, caveolin-1 also associates with LD⁽Reference Fujimoto, Kogo and Ishiguro^75–Reference Brasaemle, Dolios and Shapiro⁷⁸⁾, hinting at a role for caveolin-1 in lipolysis. Accordingly, caveolin-1-deficient mice display a blunted response to pharmacological and physiological lipolytic stimuli⁽Reference Cohen, Razani and Schubert⁷⁹⁾. Surprisingly, PKA activity is not impaired in this genotype, but rather increased⁽Reference Cohen, Razani and Schubert⁷⁹⁾ as a result of the absence of aromatic residues within the caveolin scaffolding domain that mediate PKA inhibition⁽Reference Razani, Rubin and Lisanti⁸⁰⁾. Despite accentuated PKA activity, PLIN phosphorylation is dramatically reduced in the absence of caveolin-1. A likely explanation has arisen from the in vivo and in vitro evidence that caveolin-1 facilitates the interaction between the catalytic subunit of PKA and PLIN⁽Reference Razani, Rubin and Lisanti⁸⁰⁾. The heavy representation of caveolae on plasma membranes therefore suggests an important pro-lipolytic function for caveolin-1, via PLIN phosphorylation. Importantly, the contribution of caveolin-1 in the regulation of lipolysis has yet to be explored in human fat cells but certainly warrants attention.

Fatty acid-binding protein 4

FABP4, also known as adipocyte lipid-binding protein, belongs to the large family of lipid-binding proteins. This low-molecular-mass soluble protein is highly expressed in WAT and displays a high affinity for hydrophobic species such as NEFA and retinoic acids⁽Reference Bernlohr, Doering and Kelly^81, Reference Matarese and Bernlohr⁸²⁾. FABP are thought to provide solubility to NEFA and facilitate their intracellular trafficking between metabolic enzymes and membranes⁽Reference Coe and Bernlohr^83, Reference Hertzel and Bernlohr⁸⁴⁾. FABP4 physically binds to HSL in vitro and in vivo. The first 300 amino acids of HSL provide a docking domain for FABP4⁽Reference Shen, Sridhar and Bernlohr²⁰⁾. HSL and FABP4 bind 1:1 in the cytosol in response to accentuated lipolysis⁽Reference Jenkins-Kruchten, Bennaars-Eiden and Ross⁸⁵⁾. As demonstrated by fluorescence resonance energy transfer analysis, this complex translocates to LD on PKA activation⁽Reference Smith, Sanders and Thompson⁸⁶⁾.

Comparative gene identification 58

CGI-58, also known as α/β-hydrolase domain-containing protein 5, is yet another protein associated with LD. CGI-58 is a α/β-hydrolase fold-containing protein that resembles a lipase⁽Reference Schrag and Cygler⁸⁷⁾. However, the putative catalytic triad of CGI-58 contains an asparagine in place of the usual serine residue. CGI-58 in itself therefore lacks lipase activity. In the mouse CGI-58 is highly expressed in WAT and testes, and to lower levels in liver, skin, kidney, heart, stomach, and lung⁽Reference Subramanian, Rothenberg and Gomez⁸⁸⁾. CGI-58 stimulates lipolysis by potently and selectively activating ATGL⁽Reference Lass, Zimmermann and Haemmerle⁸⁹⁾. In mature murine adipocytes CGI-58 is localized to the surface of LD via association with PLINA⁽Reference Subramanian, Rothenberg and Gomez^88, Reference Yamaguchi, Omatsu and Matsushita⁹⁰⁾. On β-AR stimulation CGI-58 is rapidly dispersed to the cytosol, an event reversible with the addition of β-AR antagonists. Under these conditions CGI-58 and ATGL co-localization is greatly accentuated and tends to migrate to small LD⁽Reference Granneman, Moore and Granneman⁶⁴⁾. Interestingly, CGI-58 has recently been found to exert lysophosphatidic acid acyltransferase activity⁽Reference Ghosh, Ramakrishnan and Chandramohan⁹¹⁾. This activity is independent of its functions as an activator of ATGL. Thus, while CGI-58 overexpression in yeast increases overall phospholipid content, it reduces neutral lipid content.

In human subjects CGI-58 has been identified as a causal gene of the Chanarin-Dorfman syndrome, a disorder characterized by the accumulation of abnormally large amounts of LD in several organs⁽Reference Lefevre, Jobard and Caux⁹²⁾. In total, nine mutations of CGI-58 have been identified in patients with Chanarin-Dorfman syndrome⁽Reference Lefevre, Jobard and Caux^92, Reference Akiyama, Sawamura and Nomura⁹³⁾. CGI-58 mutants with Chanarin-Dorfman syndrome point mutations are not recruited to LD as expected and display weak interactions with PLIN⁽Reference Yamaguchi, Omatsu and Matsushita⁹⁰⁾. This outcome may be physiologically relevant to basal and PKA-stimulated lipolysis. Recently, the importance of CGI-58 in both basal and PKA-stimulated lipolysis has been shown in human adipocytes. Gene silencing of CGI-58 not only reduces basal lipolysis by half but also completely abrogates PKA-stimulated lipolysis in hMADS adipocytes (a human white adipocyte model)⁽Reference Bezaire, Mairal and Ribet³⁸⁾. The precise whole-cell dynamics involving CGI-58, PLINA and ATGL in basal and PKA-stimulated lipolysis have not been fully elucidated but CGI-58 appears important in both states.

Lipolysis and re-esterification

Attention in WAT metabolism thus far has been mainly directed towards catabolic pathways but WAT mass is also dependent on NEFA esterification. Lipolysis and esterification are not limited to fasted and postprandial states respectively, but rather undergo constant cycling in both anabolic and catabolic states⁽Reference Newsholme⁹⁸⁾. In postprandial states glucose is the main source of the glycerol backbone. The abundance of both NEFA and glucose facilitates esterification. In catabolic states glucose levels cannot support esterification; rather, phosphoenolpyruvate carboxykinase provides glycerol backbones from pyruvate via the glyceroneogenesis pathway (for review, see Forest et al. ⁽Reference Forest, Tordjman and Glorian⁹⁹⁾). Accordingly, phosphoenolpyruvate carboxykinase expression and activity are increased with fasting⁽Reference Reshef, Hanson and Ballard¹⁰⁰⁾ and β-AR agonist treatment⁽Reference Franckhauser, Antras-Ferry and Robin¹⁰¹⁾, both highly catabolic states.

Re-esterification is the esterification of NEFA on existing acylglycerol molecules. Similarly to esterification, re-esterification occurs concurrently with lipolysis⁽Reference Vaughan^102–Reference Hammond and Johnston¹⁰⁴⁾. The regulation of re-esterification is unclear. Strong correlations between re-esterification and lipolysis rates over a wide range of lipolytic flux have been observed in mature adipocytes⁽Reference Vaughan¹⁰²⁾ and a human adipocyte cell line⁽Reference Bezaire, Mairal and Ribet³⁸⁾. In hMADS adipocytes altering lipase content quantitatively changes lipolysis and re-esterification fluxes, the coupling of the two variables remaining constant and elevated at 86%⁽Reference Bezaire, Mairal and Ribet³⁸⁾. In human subjects re-esterification is estimated at 50–75%⁽Reference Reshef, Olswang and Cassuto^105, Reference Wang, Zang and Ling¹⁰⁶⁾ but can decrease to 20–35% with fasting and exercise⁽Reference Coppack, Frayn and Humphreys^107, Reference Wolfe, Klein and Carraro¹⁰⁸⁾.

It was previously thought that re-esterification of NEFA occurs through an extracellular route⁽Reference Edens, Leibel and Hirsch¹⁰⁹⁾. With current knowledge of LD structure, questions relating to trafficking dynamics extend beyond NEFA. They also apply to acylglycerol species that are synthesized in association with the smooth endoplasmic reticulum but stored and hydrolysed in LD. Preferential hydrolysis or esterification of one acylglycerol species over another is therefore of interest. It has previously been shown that DAG are preferentially hydrolysed over TAG during PKA-stimulated lipolysis⁽Reference Edens, Leibel and Hirsch¹¹⁰⁾. Despite overall activation of lipolysis, this preferential hydrolysis occurs because of the strong capacity and affinity of HSL for DAG in human WAT⁽Reference Bezaire, Mairal and Ribet^38, Reference Mairal, Langin and Arner⁴¹⁾. Conversely, it has been found that DAG are preferentially re-esterified in the basal state and crucial to the preservation of a fixed fractional re-esterification rate in hMADS adipocytes. While forskolin uncouples re-esterification from lipolysis, inhibition of HSL restores the coupling⁽Reference Bezaire, Mairal and Ribet³⁸⁾. The implication of these findings in human adipocytes could be favoured re-esterification in obese individuals, for whom PKA-activated DAG breakdown by HSL is challenged⁽Reference Jensen, Haymond and Rizza^3, Reference Langin, Dicker and Tavernier^40, Reference Large, Reynisdottir and Langin¹¹¹⁾.

Lipolysis and adipose tissue inflammation

The past 15 years have provided evidence of the endocrine function of WAT. WAT secretes numerous proteins implicated in the control of energy homeostasis, blood pressure and coagulation, vasculature and the immune system. Immune system proteins are not only intrinsically produced and secreted by adipocytes but also by WAT-resident macrophages. As adiposity increases, so does WAT infiltration of macrophages⁽Reference Weisberg, McCann and Desai^112, Reference Xu, Barnes and Yang¹¹³⁾. WAT-resident macrophages express and secrete pro-inflammatory factors and establish the low-grade inflammation state observed in WAT with obesity and believed to be an important mediator of insulin resistance⁽Reference Xu, Barnes and Yang^113, Reference Hotamisligil, Shargill and Spiegelman¹¹⁴⁾. FABP are involved in linking WAT inflammation and systemic effects. Targeting FABP with a small-molecule inhibitor reduces WAT macrophage infiltration and the expression of inflammatory products by macrophages⁽Reference Furuhashi, Tuncman and Gorgun¹¹⁵⁾. Moreover, FABP deficiency in either macrophages or adipocytes improves insulin action and signalling⁽Reference Furuhashi, Fucho and Gorgun¹¹⁶⁾. This process is thought to occur as a consequence of a unique lipid profile in FABP-null mice⁽Reference Cao, Gerhold and Mayers¹¹⁷⁾.

A selected group of pro-inflammatory cytokines directly promote lipolysis. The resulting elevated circulating levels of NEFA further aggravate insulin resistance. TNFα is a pro-inflammatory cytokine highly expressed in obesity. Chronic TNFα treatment induces a process termed adipocyte de-differentiation, whereby PPARγ expression levels are drastically reduced⁽Reference Zhang, Berger and Hu¹¹⁸⁾. Consequently, expression of its target genes is reduced, including HSL⁽Reference Sumida, Sekiya and Okuda¹¹⁹⁾ and ATGL⁽Reference Kralisch, Klein and Lossner^120, Reference Kim, Tillison and Lee¹²¹⁾. However, TNFα exerts pro-lipolytic effects independently of lipase content. First, TNFα interferes with the anti-lipolytic action of insulin. Specifically, TNFα inhibits insulin receptor substrate 1 activation by promoting its serine phosphorylation through the p42-44 mitogen-activated PK pathway⁽Reference Engelman, Berg and Lewis^122, Reference Fujishiro, Gotoh and Katagiri¹²³⁾. Second, TNFα increases stimulatory GTP-binding protein-coupled receptors:inhibitory GTP-binding protein-coupled receptors by markedly reducing the protein content of all three inhibitory GTP-binding protein subtypes on fat cells⁽Reference Gasic, Tian and Green¹²⁴⁾. Although this effect is limited to rodent fat cells⁽Reference Ryden, Arvidsson and Blomqvist¹²⁵⁾, TNFα-induced degradation of inhibitory GTP-binding proteins by the proteasomal pathway⁽Reference Botion, Brasier and Tian¹²⁶⁾ mitigates the anti-lipolytic action of adenosine. Last, TNFα treatment reduces total PLINA content in adipocytes⁽Reference Zhang, Halbleib and Ahmad^30, Reference Souza, Vargas and Yamamoto¹²⁷⁾ and their phosphorylation by PKA⁽Reference Souza, Palmer and Kang¹²⁸⁾. This effect promotes lipolysis by increasing exposure of lipids to ATGL and HSL.

IL-6 is a pro-inflammatory cytokine heavily secreted from visceral WAT⁽Reference Fontana, Eagon and Trujillo¹²⁹⁾. Its expression is elevated in patients suffering from obesity and type 2 diabetes⁽Reference Bastard, Jardel and Bruckert^130, Reference Vozarova, Weyer and Hanson¹³¹⁾. IL-6 stimulates basal⁽Reference Petersen, Carey and Sacchetti¹³²⁾ and PKA-activated lipolysis⁽Reference Päth, Bornstein and Gurniak¹³³⁾ and induces insulin resistance⁽Reference Rotter, Nagaev and Smith^134, Reference Lagathu, Bastard and Auclair¹³⁵⁾. Stimulation of lipolysis is thought to take place independently of PKA, through the extracellular signal-regulated kinase pathway, resulting in diminished PLINA content⁽Reference Yang, Ju and Zhang¹³⁶⁾. However, IL-6 also promotes fatty acid oxidation⁽Reference Petersen, Carey and Sacchetti^132, Reference van Hall, Steenberg and Sacchetti¹³⁷⁾ via the AMPK pathway⁽Reference Al-Khalili, Bouzakri and Glund^138, Reference Carey, Steinberg and Macaulay¹³⁹⁾. Thus, despite the pro-inflammatory status of IL-6, its overall systemic effects have been rather challenging to discern⁽Reference Kristiansen and Mandrup-Poulsen^140, Reference Carey, Petersen and Bruce¹⁴¹⁾. Conversely, the action of IL-1β is better defined. IL-1β stimulates lipolysis in cultured adipocytes⁽Reference Feingold, Doerrler and Dinarello^142, Reference Doerrler, Feingold and Grunfeld¹⁴³⁾ and inhibits lipogenesis in bone marrow adipocytes⁽Reference Delikat, Galvani and Zuzel¹⁴⁴⁾. These effects are thought to partially occur as a result of impaired phosphorylation of insulin receptor substrate 1⁽Reference Lagathu, Yvan-Charvet and Bastard¹⁴⁵⁾.

While certain pro-inflammatory cytokines stimulate lipolysis, products of lipolysis have been shown to mediate inflammation in adipose tissue⁽Reference Suganami, Nishida and Ogawa¹⁴⁶⁾. Using co-cultures of adipocytes and macrophages it has been demonstrated that saturated NEFA can activate macrophages and lead to the up-regulation of macrophage-related genes⁽Reference Suganami, Tanimoto-Koyama and Nishida¹⁴⁷⁾. Saturated NEFA can therefore be defined as adipocyte-derived paracrine mediators of WAT inflammation. This response is thought to take place through the mitogen-activated PK and NF-κB pathways⁽Reference Suganami, Tanimoto-Koyama and Nishida^147, Reference Lee, Sohn and Rhee¹⁴⁸⁾. Thus, the presence of a paracrine loop between adipocytes and macrophages probably aggravates adipose tissue inflammation. The existence of a cross talk between adipocyte fat metabolism and macrophage activation is supported by in vivo clinical data on the regulation of WAT gene expression during a dietary weight-loss programme⁽Reference Capel, Klimcakova and Viguerie¹⁴⁹⁾.