Effect of Physical Exercise on Lipolysis in White Adipocytes

J Phys Fitness Sports Med, 1(2): 351-356 (2012)

JPFSM: Short Review Article Effect of physical exercise on lipolysis in white adipocytes

Junetsu Ogasawara1*, Takuya Sakurai1, Takako Kizaki1, Kazuto Takahashi2, Hitoshi Ishida2, Tetsuya Izawa3, Koji Toshinai4, Norihiko Nakano5 and Hideki Ohno1

1 Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine,6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan 2 Third Department of Internal Medicine, Kyorin University, School of Medicine,6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan 3 Department of Sports Biochemistry, Faculty of Health and Sports Science, Doshisha University, Tataramiyakodani, Kyotanabe, Kyoto 610-0394, Japan 4 Neurology, Respirology, Endocrinology, and Metabolism, Division of Internal Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan 5 Aino Institute of Regeneration and Rehabilitation, Aino University, 4-5-4 Higashiohara, Ibaraki, Osaka 567-0012, Japan

Received: April 27, 2012 / Accepted: June 12, 2012

Abstract Fatty acids are derived from the hydrolysis of triacylglycerol (TG) found in white adipose tissue, muscle tissue and circulating lipoproteins. The mobilization of free fatty acids (FFA) from white adipose tissue contributes to about 50% of the FFA utilized during moderate- intensity exercise. The delivery of FFA from white adipose tissue is improved by hormone- stimulated lipolytic events in white adipocytes (WA). Thus, the lipolysis in WA that provides fuel for metabolism has been a highly conserved function throughout the course of evolution. This short review outlines our current understanding of the molecular regulation of TG lipases via the lipolytic cascade in WA, as well as provides an account of our recent findings concerning changes in the lipolytic molecules of WA that result from acute and habitual exercise. Keywords : Lipolysis, HSL, ATGL, perilipin 1, CGI-58, white adipocyte

In this short review, the known regulatory mechanism(s) Introduction of lipolytic responses in WA, such as the lipolytic cas- The central contribution of white adipocytes (WA) to cade and the molecular behavior of lipases and regulatory the regulation of energy homeostasis is due to the storage proteins, are outlined. Then, the authors’ recent findings and cleavage capacity of lipids as well as to their function are introduced concerning exercise-induced molecular as endocrine organs that secrete many adipokines.1) Li- changes in lipolytic factors in WA. polysis is a catabolic event involving stored intracellular triacylglycerol (TG) that releases free fatty acids (FFA) Role of the lipolytic cascade in the control of lipolysis and glycerol to provide fuel for whole-body metabolism. Thus, any alteration of lipolysis in adipocytes has an im- Lipolysis in WA is regulated by a multifaceted phenom- portant effect on energy partitioning under environmental enon that is subject to distinct temporal controls, includ- conditions. Indeed, lipolysis in WA is known to be activat- ing physical exercise. Hormonal activation of lipolysis in ed by environmental changes, such as nutrient depletion adipocytes is known to be mediated via a cAMP-depen- (fasting) or an increase in energy demand (e.g., exercise). dent signal transduction process3,4) (Fig. 1). The stimula-

While the cellular bases of the dynamic hydrolysis tion of G-protein coupled receptors (GPCRs), i.e., β1-, action of TG are well documented, the process of eluci- β2- and β3-adrenergic receptors, induces a conformational dating the molecular factors regulating these processes change in the Gα subunit of the heterotrimeric G protein continues to evolve. It is widely accepted that hierarchical (Gαβγ) that leads to GDP release and GTP binding. Acti- action via alteration in intracellular cAMP is the mecha- vated Gαs leads to the activation of adenylyl cyclase (AC) nism by which hormone-stimulated and non-hormone- and production of cAMP. On the other hand, stimulation 5) stimulated lipolysis in WA (i.e., lipolytic cascade) proof GPCRs, i.e., α2-drenergic receptor, adenosine recep- ceeds. However, recent studies have indicated that the tor,6) and prostaglandin E2 receptor,7) which stimulate trafficking of lipases and regulatory proteins is essential Gαi, causes the inactivation of AC and reduces the pro- for full lipolysis as final steps in the lipolytic cascade.2) duction of cAMP, resulting in an attenuation of the lipolytic response. In addition, insulin attenuates intracellular *Correspondence: [email protected] cAMP production through increases in phosphodiester- 352 JPFSM: Ogasawara J, et al.

Stimulatory actions E1Ͳ,E2Ͳ,E3ͲAR FA Glycerol GsD GE GJ GTP GsD GDP MGL AC Mg䊶ATP P MG GiD HSL Translocation cAMP FA GiD GE P DG FA PDEͲ3B PKA GJ HSL TG PKB/AKT P Perilipin1 ATGL Inhibitory actions CGIͲ58 PI3ͲK Lipiddroplet D2ͲAR IRSͲ1 AdenosineͲR NicotinicacidͲR Nucleus InsulinͲR Figure1

Fig. 1 The lipolytic cascade for white adipocytes Lipolysis in white adipocytes is regulated by diverse stimulatory and inhibitory actions through GPCRs that exist on the plasma membrane. Ligand binding to stimulatory GPCRs provokes AD activation via the action of Gsα, resulting in the PKA-mediated phosphorylation of lipase and regulator proteins, and thereby the activation of lipolysis. On the other hand, insulin and inhibitory ligands attenuate lipolysis via a reduction in the cAMP-mediated activation of PKA. -AR, adrenergic receptor;-R, receptor; PKA, cAMP-dependent protein kinase A; TG, triacylglycerol; DG, diacylglycerol; MG, monoacylglycerol; IRS-1, insulin receptor substrate-1;PI3-K, phosphatidil inositol 3 kinase; PKB, PDE-3B, phophodiesterase-3B; MGL, monoglyceride lipase.

ase-3B (PDE-3B) activity, which changes cAMP to AMP glycerol (DG) and monoacylglycerol (MG) and glycerol. via activation of protein kinase B/AKT. An increased HSL exhibits DG hydrolase activity that is 10- and 5-fold intracellular cAMP level phosphorylates and activates greater than its activity against TG and MG, respec- cAMP-dependent protein kinase A (PKA),8,9) which, in tively,14) and is considered to be the major lipase mediat- turn, phosphorylates hormone-sensitive lipase (HSL), a ing hormone-stimulated lipolysis in adipocytes.15) HSL rate-limiting enzyme of lipolysis, and activates hydrolysis activation is regulated by reversible phosphorylation by of TG in adipocytes10) via the translocation of HSL on the protein kinases. The phosphorylation of HSL at Ser563, lipid droplet surface.11) Under activation of PKA, perilipin Ser659 and Ser660, by cAMP-dependent protein kinase 1, which is a dominant target of PKA activation,12) is also (PKA), is known to enhance its enzymatic activity; and phosphorylated and acts as a scaffold protein, resulting in extracellular-regulated kinase (ERK) increases HSL activ- facilitated lipolysis by allowing the subcellular localiza- ity via phosphorylation at Ser600 in 3T3-L1 adipocytes,16) tion of HSL to lipid droplets.13) Taken together, lipolytic although this has not been observed in either human or responses provide a conceptual network that is regulated murine primary adipocytes. On the other hand, insulin by the PKA-mediated trafficking of phosphorylated pro- has an inhibitory effect on the cAMP-dependent and -in- teins to lipid droplet surfaces. dependent activation of HSL.17,18) AMP activated protein kinase (AMPK) is known to attenuate the enzymatic activity of HSL through an increase in the phosphorylation Lipase control of lipolysis via the sequential hydrolysis of HSL at Ser565.19) In addition, glycogen synthase ki- of TG nase-420) and Ca2+/calmodulin-dependent kinaseⅡ21) have The hydrolysis of TG in adipocytes is regulated by also been reported to cause phosphorylation of HSL at specific enzymes, and results in the liberation of fatty Ser563 and Ser565, respectively. Thus, the phosphoryla- acids (FA) at several steps, with the production of diacyl- tion of HSL is known to play a central role in the regula- JPFSM: Regulation of lipolysis in white adipocytes 353 tion of enzyme activity, and is closely associated with the the enzymatic activity of HSL. However, the activation of catabolism of adipocytes. PKA in response to β-AR causes up to a 100-fold induc- Until recently, HSL was considered to be the only, and tion of FA and glycerol release in intact adipocytes, in- therefore the rate-limiting, enzyme for the lipolytic ca- dicating the possibility that other mechanisms contribute tabolism of intracellular TG in adipocytes and nonadipose to hormone-induced lipolysis. The discovery of perilipin cells, because of its capacity to hydrolyze both TG and 1 provided proof that this was indeed the case.30) Perili- DG as substrates. However, studies focusing on white pin 1 is expressed mostly in white adipose tissue and in adipocytes of HSL knockout mice have revealed that no steroidogenic tissue.12) Perilipin 1 can be phosphorylated complete loss of activity in the hydrolysis of TG occurs at as many as six sites (Ser81, Ser222, Ser276, Ser433, in mice adipocytes,22-24) which indicates that, in addition Ser492 and Ser517) by PKA31-34) and is a major target of to HSL, other lipases are involved in the TG degradation PKA phosphorylation in adipocytes.12) Previous studies of adipocytes. In 2004, three groups independently pub- have demonstrated that perilipin 1 is a multifunctional lished the discovery of an enzyme that could hydrolyze protein that is capable of reducing basal lipolysis by TG,25-27) and Zimmermann et al. named it adipose triglyc- combining HSL with lipid droplets to form a barrier,35) eride lipase (ATGL). ATGL has a 10-fold higher substrate promotes the lipolysis movement of perilipin 1 away specificity for TG than for DG, and it selectively acts as from fat droplets,36) and controls lipid droplet fragmenta- the first step in TG hydrolysis, resulting in the formation tion through lipase-dependent and -independent mecha- of DG and FA.25) Interestingly, unlike HSL, ATGL has no nisms.37) Moreover, the recent comparative gene identifi- specificity for the hydrolysis of MG, cholesteryl esters, or cation-58 (CGI-58), also known as α/β hydrolase domain- retinyl esters, demonstrating that its hydrolytic function is containing protein 5 (ABHD5), is known to increase the not restricted to the catabolism of lipid droplets28) in adi- TG hydrolase activity of ATGL due to direct interaction pose tissue. In addition, two phosphorylation sites, Ser404 with ATGL proteins.38) Interestingly, CGI-58 has the abil- and Ser428, of ATGL were identified in the C-terminal ity to associate with perilipin 1.39-41) The molecular behav- region in humans.25,29) However, in contrast to HSL, the iors of both perilipin 1 and CGI-58 are centrally involved functional roles of enzyme phosphorylation, as it involves in the organization and regulation of lipolytic effector protein kinases, remain unknown. Taken together, both interactions in both basal and stimulated states. ATGL and HSL act hierarchically to regulate TG hydro- The recent consensus concerning the mechanisms lysis: ATGL initiates lipolysis by removing the first FA underlying the regulation of lipolysis in adipocytes is from TG to, in turn, produce DG; and HSL generates an described below (Fig. 2). Under basal conditions, ATGL additional FA from DG and MG to produce glycerol. is localized mainly within the cytoplasm, although CGI- 58 exists on the surface of lipid droplets and interacts with perilipin 1,42) thereby lowering the levels of ATGL- Role of lipolytic factors coupled with lipases CGI-58 interaction. HSL is located almost entirely in the Phosphorylation and activation of HSL by PKA in re- cytoplasm, where it is removed from lipid droplets.12) In sponse to β-AR stimuli induces a moderate increase in contrast, β-AR stimulation causes CGI-58 to move into

A Basal and inactivated conditions BHormone-activated condition

Lipid droplet 䋨TG䋩 Lipid droplet 䋨TG䋩 Pe Pe rili ATGL rili ATGL p p P in

P P

i L C n P 1 CGI-58 G 1 P Perilipin 1 I- P P P 5 HS 8 Dissociation P P P P P P P Perilipin 1 Binding P P P Release HSL ATGL HSL CGI-58 Translocation

PKA Cytoplasmic space Cytoplasmic space

Fig. 2 The regulation of lipolysis by lipases (A) Basal state: perilipin 1 and CGI-58 form a complex on the lipid droplet. (B) Activated state: phosphorylation of perilipin 1 by PKA releases CGI-58, which binds ATGL to induce lipolysis. 354 JPFSM: Ogasawara J, et al. the cytoplasm, which is mediated within minutes42) by the pocytes, such as AKAP150, CGI-58 and perilipin 1, and dissociation of phosphorylated perilipin 1 at Ser517 and PPAR-γ2. CGI-58.32) After dissociation of perilipin 1, CGI-58 asso- However, the adaptation of adipocytes in response to ciates with ATGL in fragmented lipid droplets and at sites habitual exercise training appears to be the result of the lacking perilipin 1.42,43) At this time, PKA promotes the integrative effect of acute exercise. Thus, it is important translocation of phosphorylated HSL at Ser659 and 660 to investigate the effect of acute exercise on lipolysis in from the cytoplasm to lipid droplets,44) resulting in an in- adipocytes in order to strengthen and clarify understand- ducement of maximal lipolytic response. Thus, the results ing of the molecular mechanisms underlying chronic of previous studies demonstrate that the phosphorylation exercise-induced adaptive modulation of adipocytes. In- and trafficking of lipases and lipolytic proteins play a deed, it is shown that the function of β2-AR is upregulated critical role in regulating basal and hormone-stimulated by acute exercise in WA.49) Similar to the results of habit- lipolysis in adipocytes. ual exercise, glycerol release in primary adipocytes was significantly elevated immediately (0 h) and three hours (3 h) after acute exercise with increases in both activity Manipulation of lipolytic molecules by active exercise (phosphorylation at Ser563 and Ser660) and localiza- in supplying energy tion of HSL to the lipid droplets. Under these conditions, An understanding of the regulatory mechanisms un- although neither perilipin 1 nor CGI-58 protein levels derlying basal and hormone-stimulated lipolysis in adi- changed, the level of perilipin 1/CGI-58 complex was pocytes has evolved in recent years. However, little is significantly reduced, accompanied by an upregulated as- known about the effect of acute and/or habitual physical sociation of perilipin 1/HSL for up to 3 h after acute exer- exercise on the molecular behavior of lipolytic proteins, cise, indicating that acute exercise enhances lipolysis in a i.e., HSL, ATGL, perilipin 1 and CGI-58, in white adipo- manner that is dependent on the modification of HSL and cytes. Almost 30 years have passed since a pharmacologi- its association with, and alteration of, CGI-58 and perili- cal approach was used to first report that acute and habit- pin 1 proteins.50) ual physical exercise in rats enhanced lipolytic responses A summary of the authors’ studies, which focused only in both adipose tissue and isolated adipocytes.45,46) It is on the molecular changes in HSL, ATGL, CGI-58, perili- important to note that the major lipolytic alteration in- pin 1 and AKAP150, is shown in Table 1. These results duced by exercise training is documented as occurring at indicate that acute exercise-enhanced lipolysis is regu- a site distal to hormonal regulation of the β-AR, although lated not only by newly synthesized proteins, but also by no differences have been found in intracellular cAMP post-translational modification events, such as protein accumulation compared with a sedentary control,47) sug- interactions. Conversely, habitual exercise increases lev- gesting the possibility that the molecular behavior of els of lipases with transcriptional upregulation, suggest- lipolytic proteins plays a key role in the exercise training- ing that transcriptional factors play a critical role in these induced alteration of lipolysis. To this end, the authors adaptive changes. However, quite recently it has been un- investigated the possible mechanisms behind exercise derstood that habitual exercise induces no change in the training-enhanced lipolysis in rat primary adipocytes.48) levels of perilipin 1 and CGI-58 proteins, although levels Results showed that exercise training enhanced the levels of the interaction partners of these proteins, i.e., HSL and of catalytic subunits of PKA (RIIα, and RIIβ) proteins ATGL, are significantly elevated. and PKA-anchoring protein 150 (AKAP150), which sup- ports the binding of PKA and its substrate, as well as the activity of both PKA and HSL in the lipid droplet frac- Table 1. Effect of exercise on levels of proteins and interactions. tion of adipocyte homogenate. These results suggest that Primary white adipocytes in rats (epididymal) exercise training provokes an increase in the anchoring of AKAP150 to PKA, thereby augmenting the magnitude Acute exercise Habitual exercise of cAMP signaling, even if accumulations of intracellular (Levels of protein) cAMP fail to increase. Moreover, the authors recently Perilipin 1 㫧 no change Perilipin 1 㫧 no change observed that exercise training-enhanced lipolysis was CGI-58 㫧 no change CGI-58 㫧 no change closely associated with the upregulation of ATGL and HSL 㫧 no change HSL 䋫 increase DNA-binding activities of peroxisome proliferation-ac- ATGL 㫧 no change ATGL 䋫 increase tivated receptor-γ2 (PPAR-γ2); and that binding of CGI- AKAP150 䋿 unknown AKAP150 䋫 increase 58 to ATGL, with dissociation of CGI-58 and perilipin 1, significantly increased on the lipid droplets of primary (Protein interactions) adipocytes (unpublished data). Therefore, the mechanisms HSL / perilipin 1 䋫 increase HSL / perilipin 1 䋫 increase behind exercise training-enhanced lipolysis in adipocytes ATGL / CGI-58 䋿 unknown ATGL / CGI-58 䋫 increase include the upregulation of both HSL and ATGL, which CGI-58 / perilipin 1 䋭 decrease CGI-58 / perilipin 1 䋭 decrease are modified by the molecular behavior expressed in adi- JPFSM: Regulation of lipolysis in white adipocytes 355

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