Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A Academic research paper on "Biological sciences"

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Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A^

Harald M. Nagy1, Margret Paar1, Christoph Heier, Tarek Moustafa, Peter Hofer, Guenter Haemmerle, Achim Lass, Rudolf Zechner, Monika Oberer, Robert Zimmermann *

Institute of Molecular Biosciences, University of Graz, Austria

ARTICLE INFO

ABSTRACT

Article history:

Received 7 November 2013

Received in revised form 20 December 2013

Accepted 6January2014

Available online xxxx

Keywords:

Adipose triglyceride lipase

Hormone-sensitive lipase

Lipolysis

Regulation

acyl-CoA

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycer-ides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the proteinprotein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites.

© 2014 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction

Adipose triglyceride lipase (ATGL, also referred to as patatin-like phospholipase domain containing 2 [PNPLA2] or desnutrin [1]) performs the first step in triglyceride (TG) hydrolysis generating diglyceride (DG) and free fatty acids (FFAs) [2]. Consequently, the enzyme controls the availability of FFAs, which may serve as energy substrates, precursors for other lipids, and lipid signaling molecules. This central function has a major impact on overall energy metabolism and becomes evident in fasted ATGL-deficient mice (ATGL-ko). In this mouse model, the lack of sufficient FFAs for energy conversion promotes the usage of glucose for energy conversion [3]. As a consequence, short fasting periods or moderate exercise leads to rapid consumption of glycogen stores. Fasting for more than 6 h results in hypoglycemia, hypometabolism, and hypothermia [3,4].

ATGL activity is regulated by a complex network of hormones which control enzyme expression and the interaction of the enzyme with the

☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Corresponding author at: Institute of Molecular Biosciences, University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria.Tel.: +43 316 3801900; fax: +43 316380 9016.

E-mail address: robert.zimmermann@uni-graz.at (R. Zimmermann).

1 Authors contributed equally.

regulatory proteins. ATGL is stimulated by the presence of an activator protein as observed for other TG lipases, such as pancreatic lipase or li-poprotein lipase. The activator of ATGL is termed comparative gene identification-58 (CGI-58) [or alpha/beta-hydrolase domain containing 5 (ABHD5)] [5]. Currently, the molecular mechanism on how CGI-58 stimulates ATGL activity is unknown [6]. However, loss of either ATGL or CGI-58 function causes systemic TG accumulation in humans and mice. This inherited disorder is known as Neutral Lipid Storage Disease (NLSD) [7]. A second regulatory protein of ATGL is G0/G1 switch gene-2 (G0S2). This protein was originally described to be required to commit cells to enter the G1 phase of the cell cycle [8]. Recent evidence suggests that G0S2 specifically inhibits ATGL activity in rodents and humans [9,10]. Both G0S2 and CGI-58 have been shown to interact with ATGL. Furthermore, they are present on lipid droplets and regulated by metabolic hormones. G0S2 appears to be regulated primarily on the expression level. The antilipolytic hormone insulin increases G0S2 expression in 3T3-L1 adipocytes, whereas activation of lipolysis by fasting, (3-adrenergic agonists, and tumor necrosis factor-a has the opposite effect [9,11]. In contrast to G0S2, fasting and (3-adrenergic stimulation have minor effects on CGI-58 protein expression in adipose tissue. This co-activator protein is regulated primarily by its reversible interaction with the lipid droplet coating protein perilipin 1 [12]. In non-activated adipocytes, CGI-58 is bound to perilipin 1 and lipolysis is low. Upon lipolytic stimulation by (3-adrenergic agonists, perilipin 1 gets phosphory-lated by protein kinase A leading to the release of CGI-58 which is

1388-1981/$ - see front matter © 2014 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.! 016/j.bbalip.2014.01.005

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now available for ATGL activation. In addition, ATGL activity is influenced by other members of lipid droplet coat proteins of the perilipin (PAT) family. Perilipin 2 has been shown to reduce the lipid droplet association of ATGL [13]. Recent data also suggest that perilipin 5 interacts with ATGL and inhibits its activity [14-17].

Up to date, it was unknown whether ATGL activity or its interaction with regulatory proteins is controlled by lipid metabolites arising during lipolysis. Here we show that ATGL is directly inhibited by long-chain acyl-CoA via a non-competitive mechanism.

2. Materials and methods

2.1. Materials

Acyl-CoA with various fatty acid chain lengths and triolein were obtained from Sigma-Aldrich (Taufkirchen, Germany). Radiolabeled [9,10(N)-3H]triolein was obtained from PerkinElmer Life Sciences and hexadecyl-CoA was obtained from Avanti Polar Lipids.

22. Expression of recombinant proteins

For expression of murine ATGL and CG1-58 in Escherichia coli, sequences containing the complete open reading frame of murine ATGL and murine CG1-58 were amplified from cDNA by PCR using Phusion ™ High Fidelity DNA Polymerase (Finnzymes, Espoo, Finland). Respective primers were designed to create 5' and 3' restriction endonuclease cleavage sites (underlined) for subsequent cloning strategies:

mATGL_fw: 5'-TCGGTACC CATGTTCCCGAGGGAGACCAA-3'

mATGL_rv: 5'-ACCTCGAG TCAGCAAGGCGGGAGGC-3'

mCGl-58_fw: 5'-GGGGATCC CAAAGCGATGGCGGCGG-3'

mCGl-58_rv: 5'-CTGATATC TCAGTCTACTGTGTGGCAGATCTCC-3'.

PCR products were inserted into the target vector pASK-lBA5plus (IBA, Goettingen, Germany) and transformed into E. coli (strain XL-1 and BL-21 for ATGL and CG1-58, respectively). Protein expression was induced by adding 200 ng/ml anhydro-tetracycline. Cells were harvested 3 h after induction. Expression of strep-tagged proteins was detected by Western blot analysis using mouse anti-Strep-tag II antibody (1:5000 dilution; IBA, Goettingen, Germany) as primary antibody and HRP-linked sheep-anti mouse antibody, (1:10,000; GE Healthcare Amersham, Buckinghamshire, UK) as secondary antibody.

Transient transfection of Monkey embryonic kidney cells (COS-7, ATCC CRL-1651) with pcDNA4/HisMax coding for His-tagged ATGL, HSL, or £-galactosidase (LacZ) was performed with Metafectene™ (Biontex GmbH) as described [2]. Expression of His-tagged proteins was detected using anti-His monoclonal antibody (6xHis, BD Biosciences) and a horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare) as secondary antibody.

23. Preparation of cell and tissue extracts

E. coli and COS-7 cells were disrupted by sonication resuspended in lysis buffer in lysis buffer (0.25 M sucrose, 1 mM dithiothreitol, 1 mM EDTA, 20 |ag/ml leupeptine, 2 ^g/ml antipain, 1 ^g/ml pepstatin, pH 7.0). Lysates of E. coli were centrifuged at 15,000 xg at 4 °C for 20 min. For the preparation of COS-7 cell extracts, nuclei and unbroken cells were removed by centrifugation at 1000 xg at 4 °C for 5 min. Su-pernatants were collected and used for activity assays. The specific activity of these lysates ranged from 100 to 400 nmol/h-mg depending on the expression levels of recombinant proteins.

Mouse gonadal WAT was homogenized in lysis buffer (~1 ml/fat pad) using an Ultra Turrax Homogenizer (Fisher Scientific, Waltham, MA). The homogenate was centrifuged at 20,000 xg at 4 °C for 1 h. The interphase was collected and used for activity assays.

2.4. Assay for TG hydrolase activity

The substrate for the measurement of TG hydrolase activity was prepared as described previously with minor modifications [2]. Briefly, triolein and [9,10-3H]triolein (10 ^Ci/ml) were emulsified in the presence of phosphatidylcholine/phosphatidylinositol using a sonicator (Virsonic 475, Virtis, Gardiner, NJ) and adjusted to 2.5% BSA (FFA free). The final substrate concentration was 1.67 ^mol/ml triolein and 0.15 mg/ml PC/PI (3:1). For kinetic investigations, the TG substrate was diluted to the indicated concentrations after sonication. Activity assays were performed using 0.1 ml ofcell lysates and 0.1 ml substrate in a water bath at 37 °C for 20 min. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation at 800 xg for 20 min, the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting.

2.5. Protein interaction of ATGL and CGI-58

Cos-7 cells were co-transfected with Flag-tagged CGI-58 and His-tagged ATGL. After 3 h of binding, FLAG-beads were washed and incubated for 20 min with indicated concentrations of acyl-CoAs at 37 °C. Subsequently, beads were washed 3-times with lysis buffer, proteins were eluted by boiling in SDS-containing sample puffer, and probes were subjected to Western blot analysis using FLAG- and His-tag specific antibodies (Monoclonal mouse ANTI-FLAG® M2-Peroxidase (HRP) antibody, Sigma, A8592; Monoclonal mouse ANTI-HIS antibody, GE Healthcare, 27-4710-01) and sheep anti-mouse IgG (HRP-linked, GE Healthcare; NA931) as secondary antibody.

2.6. Protein determinations

Protein concentrations of cell lysates were determined by Bio-Rad protein assay kit according to manufacturer's instructions (Bio-Rad, Hercules, CA) using BSA as standard.

2.7. Statistical analysis

Data are presented as mean ± S.D. Statistical significance was determined by the Student's unpaired t-test (two-tailed). Group differences were considered significant for p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

3. Results

3.1. ATGL is inhibited by oleoyl-CoA

Inhibition of ATGL activity by lipid intermediates was first investigated in lysates of COS-7 cells expressing His-tagged ATGL and CGI-58. Lysates containing approximately equimolar concentrations of ATGL and CGI-58 (Fig. 1A) were incubated with a radiolabeled triolein substrate in the absence (control) or presence of various lipid metabolites. At a concentration of 50 ^M, ATGL activity was almost completely inhibited in the presence of oleoyl-CoA. In comparison, oleic acid had little effect and free CoA, (OA), monoolein (MO), and diolein (DO) did not affect enzyme activity (Fig. 1B). ATGL was also inhibited by a thioether analog of palmitoyl-CoA (hexadecyl-CoA, Fig. 1B) suggesting that protein acylation is not required for inactivation.

Addition of oleoyl-CoA led to inactivation of ATGL activity with an 1C50 value of 33 ^M (Fig. 1C).

It is important to note that all assays have been performed in the presence of excess BSA (360 ^M) which harbors high affinity sites for acyl-CoA [18]. When BSA was omitted from the reaction, we observed an almost complete inhibition of ATGL activity implicating that the enzyme requires an FFA acceptor for full activity. In the presence of low amounts of BSA (3.6 |jM), the enzyme retained ~50% of its activity.

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IC50 = 33 MM

0 5 10 20 30 40 50 100 MM Oleoyl-CoA

■— 5 % BSA - -•- - 0.05 % BSA

100 ■

80 ■

60 ■

40 ■

20 ■

/ Cß 0° JT

Fig. 1. ATGL is inhibited by oleoyl-CoA. (A) Western blot analysis of COS-7 cell lysates overexpressing ATGL and CGI-58. (B) TG hydrolase activity of ATGL in presence of 50 |jM oleoyl-CoA, free CoA, free oleic acid, rac-MO, rac-DO and a thioether analog of palmitoyl-CoA (Hexdecyl-CoA). (C) Effect of BSAon oleoyl-CoA mediated inhibition of ATGL activity. The specific activity decreased from 3.2 |mol/h-mg to 1.7 |mol/h-mg when the BSA concentration was reduced from 5% (360 |M) to 0.05%. Data are presented as mean ± S.D. from triplicate determinations and representative for at least three independent experiments.

186 Under these conditions, we determined an IC50 value of 21 |jM suggest-

187 ing that BSA moderately interferes with acyl-CoA-mediated enzyme in-

188 hibition (dashed line, Fig. 1C).

189 3.2. ATGL and HSL are inhibited by long-chain acyl-CoAs

190 HSL has previously been shown to be inhibited by long-chain acyl-

191 CoAs (LCAs) [19]. To compare the effect of acyl-CoAs of different chain

192 length on ATGL and HSL activity, we expressed these enzymes in COS-

193 7 cells and determined TG hydrolase activity in the presence of various

194 acyl-CoAs. The acyl-CoA-mediated inhibition of TG hydrolysis strongly

195 depended on fatty acid length. Both, ATGL and HSL were inhibited by

196 oleoyl-CoA and palmitoyl-CoA (Fig. 2A). Lauroyl-CoA inhibited HSL

197 but had no effect on ATGL activity. Acyl-CoA with shorter chain length

198 did not inhibit either enzyme. Next, we tested the effect of acyl-CoAs

199 in lysates of mouse white adipose tissue (WAT) where ATGL and HSL

200 are together responsible for more than 95% of the neutral TG hydrolase

201 activity [20]. In WAT lysates of wild-type and ATGL-ko mice, TG hydro-

202 lase activity was inhibited by acyl-CoAs exhibiting a fatty acid chain

203 length > 12 carbon atoms (Fig. 2B, C). In WAT lysates of HSL-deficient

204 (HSL-ko) mice, where ATGL represents the major TG lipase, TG hydro-

205 lase activity was inhibited by palmitoyl- and oleoyl-CoA (Fig. 2D)

206 whereas lauroyl-CoA had no effect. Thus, both ATGL and HSL are

207 inhibited by LCA and only HSL is sensitive to lauroyl-CoA allowing dis-

208 crimination between ATGL and HSL activity in biological samples.

209 3.3. Acyl-CoAs interact with the N-terminal domain of ATGL and do not

210 disrupt the protein interaction of ATGL and CGI-58

211 Next we investigated whether LCAs inhibit ATGL activity directly by

212 binding to the enzyme or indirectly by binding to CGI-58 and interfering

213 with ATGL/CGI-58 interaction. To test if the inhibition is dependent on

CGI-58, we omitted CGI-58 from the reaction. As shown in Fig. 3A, 214

ATGL was sensitive to LCA-mediated inhibition in the presence and ab- 215

sence of CGI-58 indicating that LCAs directly interact with the enzyme. 216

To restrict the binding site of LCAs to N- and C-terminal domains, we 217

used a truncated version of ATGL lacking the C-terminal part of the en- 218

zyme (Q289ter). This truncated ATGL variant comprises the active 219

patatin-like phospholipase domain and has previously been shown to 220

exhibit increased lipase activity in comparison to the full-length enzyme 221

[21]. As shown in Fig. 3C, Q289ter was inactivated by oleoyl-CoA which 222

suggests that LCAs interact with the N-terminal domain of ATGL com- 223

prising the catalytic patatin-like region [22]. 224

To investigate whether LCAs affect the interaction of ATGL and CGI- 225

58, we performed immunoprecipitation assays using His-tagged CGI-58 226

and FLAG-tagged ATGL. As expected, we could clearly detect an interac- 227

tion between these proteins when co-expressed in COS-7 cells (Fig. 3B). 228 Addition of oleoyl-CoA or lauroyl-CoA had no effect suggesting that 229

acyl-CoAs do not disrupt the interaction of ATGL and its activator 230

protein. 231

To exclude that other cofactors are required for LCA-mediated inhi- 232

bition of ATGL, we switched to a heterologous expression system and 233

expressed Strep-tagged ATGL and CGI-58 in E. coli. As shown in 234

Fig. 3C, ATGL was active in E. coli lysates and inactivated by the addition 235

of oleoyl-CoA with an IC50 value of 16 |M. Together, these observations 236

suggest that LCA directly inhibit ATGL. 237

3.4. Long-chain acyl-CoAs inhibit ATGL in a non-competitive manner 238

To get insight into the mechanism of inhibition, we performed in- 239

hibitor kinetic studies. For this purpose, we used E. coli lysates contain- 240

ing ATGL and CGI-58. First, we tested whether we can apply Michaelis- 241

Menten kinetics. As shown in Fig. 4A, saturation kinetics revealed an 242

almost linear increase in enzyme activity up to 800 |M substrate. 243

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COS-7 lysates

WAT WT

G G G ■ ATGL/CGI-58 DHSL

„Ä Ä * & c? ci*

WAT ATGL-ko

WAT HSL-ko

A A Cr cT

Fig. 2. ATGL and HSL are specifically inhibited by long-chain acyl-CoAs. (A) TG hydrolase activity of COS-7 cell lysates overexpressing ATGL/CGI-58 or HSL in presence of acyl-CoAs with different acyl-chain lengths. (B), (C), (D) Acyl-CoA-mediated inhibiton ofTG hydrolase activity in WATlysates ofwild-type, ATGL-ko, and HSL-ko mice, respectively. The specific activity of wild-type lysate was 412 ± 77 nmol/h-mg. TG hydrolase activities in ATGL-ko and HSL-ko samples are decreased by 65% and 72%, respectively, as described earlier [20]. Data are presented mean ± S.D. from triplicate determinations and representative for two independent experiments.

Furthermore, time course experiments demonstrated that the reaction was linear for at least 30 min in the absence and presence of oleoyl-CoA suggesting that steady-state conditions are achieved (Fig. 4B). Inhibitor kinetics were performed using different substrate and inhibitor concentrations in a concentration range of 200-800 |jM triolein and 0, 20, and 40 ^M oleoyl-CoA, respectively. As shown in Fig. 4C, inhibition of ATGL by oleoyl-CoA was almost independent of the substrate concentration. Accordingly, Lineweaver-Burk analysis revealed that oleoyl-CoA reduced Vmax but did not affect Km demonstrating that acyl-CoA-mediated inhibition occurs in a non-competitive manner (Fig. 4D). Using nonlinear regression analysis (GraphPad Prism 5, GraphPad Inc.) and a model for mixed inhibition kinetics we calculated a Ki of 19 ± 5 |JM.

4. Discussion

Generally, FFAs have to be activated to acyl-CoAs for further metabolization such as (3-oxidation, synthesis of complex lipids, or protein acylation. It is well known that acyl-CoAs are not only short-lived metabolites, but directly regulate central enzymes in energy and lipid metabolism including mitochondrial adenine nucleotide translocase, acetyl-CoA carboxylase, pyruvate dehydrogenase, and phosphofructoki-nase [23]. Furthermore, LCAs regulate the activity of different protein ki-nase C subtypes and are directly or indirectly involved in the control of gene expression, ion fluxes, and membrane trafficking [23].

Previous studies demonstrated that LCAs inhibit tissue TG lipase activity [24,25] and HSL by non-competitive inhibition [19,26]. Here we show that LCA also target ATGL and inhibit the enzyme in a non-competitive manner. Our data indicate that differences exist with respect to the efficacy of acyl-CoA species, since HSL but not ATGL is

inhibited by lauroyl-CoA. We assume that this observation does not 272 have physiological relevance, since lauric acid is a minor component 273 of cellular lipids (~0.3% in WAT; [27]). Conversely, both enzymes are 274 inhibited by acyl-CoA esterified with the highly abundant fatty acid spe- 275 cies palmitic acid and oleic acid. Since inactivation of ATGL and HSL al- 276 most completely abolishes WAT fatty acid release [20], our data 277 suggest that LCA control the activity of the major lipolytic enzymes. It 278 is important to note that FFA metabolism is causally linked to metabolic 279 disease. Increased circulating FFAs, as observed in obesity, can cause FFA 280 overload of non-adipose tissues resulting in ectopic TG accumulation 281 which is associated with impaired metabolic functions of these tissues, 282 insulin resistance, and inflammation. These changes are not caused by 283 the increase of the inert TG storage pool. It is believed that elevated cel- 284 lular FFA levels promote the synthesis of lipotoxic metabolites such as 285 ceramides, acyl-CoAs, and diacylglycerol [28-30]. FFA overload may re- 286 sult from increased lipolysis and impaired (3-oxidation [31] and both 287 processes can elevate cellular acyl-CoA concentrations. Notably, acyl- 288 CoA concentrations are increased in tissues of insulin resistant subjects 289 and the correlation between muscle acyl-CoA content and insulin resis- 290 tance is stronger than that between muscle TG stores and insulin resis- 291 tance [32]. Under such conditions, acyl-CoA-mediated inhibition of 292 lipolysis could represent a principle feedback mechanism reducing 293 FFA concentrations and promoting the storage of inert TG. 294

It is interesting to note that the increase in circulating FFA levels in 295 obese patients is modest in comparison to the enormous expansion of 296 WAT [33]. This suggests that WAT maintains its ability to control lipoly- 297 sis despite hypertrophy and hyperplasia. Actually, FFA release per kilo- 298 gram fat mass is reduced in obesity [33] and elevated plasma FFA 299 levels may result from increased adipose mass. Acyl-CoA-mediated in- 300 hibition of lipolysis may be one mechanism preventing TG degradation 301

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120 100 80 60 40 20 0

pM Oleoyl-CoA

IATGL WT DATGL Q289ter

16.6 pM

10 20 30 40 pM Oleoyl-CoA

empty FLAG FLAG-mATGL 6xHis-mCGI58 C 12:0-CoA C 18:1-CoA

+ + + + + + + + + + +

- 5pM 50pM -

- 5pM 50^M

CGI-58 ATGL

Fig. 3. Oleoyl-CoA directly interacts with the N-terminal domain of ATGL and does not affect the protein interaction of ATGL and CGI-58. (A) Oleoyl-CoA-mediated inhibiton of wild-type ATGL and the truncated Q289ter mutant without addition of CGI-58. (B) Effect of C12-CoA and C18:1-CoAon the protein-protein interaction of ATGL and CGI-58. Cos-7 cells were co-transfected with His-tagged CGI-58 and FLAG-tagged ATGL. After incubation with the lysates, FLAG-beads were incubated for 20 min with indicated concentrations of acyl-CoAs at 37 °C. After extensive washing, proteins were eluted by boiling in SDS-containing sample puffer and subjected to Western blot analysis. (C) Oleoyl-CoA-mediated inhibiton ofStrep-tagged ATGL and CGI-58 expressed in E. coli. The specific activity of these lysates ranged from 100 to 400 nmol/h- mg depending on the expression levels of recombinant proteins. Activity data are presented as mean ± S.D. from triplicate determinations and representative for three independent experiments.

302 in obese subjects and this could be specifically important in the insulin

303 resistant state, since insulin is considered as the major suppressor of li-

304 polysis [34].

305 Cellular LCA concentrations have been reported to be in the range of

306 5-160 |jM and strongly depend on the metabolic state and tissue-type

307 [35]. Accordingly, the IC50 values determined for ATGL inhibition are

308 clearly within the physiological range. However, it has to be considered

309 that LCA are primarily bound to acyl-CoA binding protein (ACBP) acting

310 as acyl-CoA transporter [36]. Additionally, liver fatty acid binding pro-

311 tein can bind LCA with high affinity [37]. Because of the high cellular

312 concentrations of LCA binding proteins, it is assumed that LCAs are pres-

313 ent in their free form in very small amounts [36]. Currently, it is unclear

314 how ACBP-bound LCA affect ATGL activity. Yet, it has been shown that

315 ACBP-bound LCA are available for metabolic or regulatory processes

316 such as beta-oxidation, synthesis of lipids, and signal transduction

317 [35]. Furthermore, addition of ACBP promotes the inhibitory effect of

318 palmitoyl-CoA on partially purified HSL [25]. It was also shown that

319 LCA stimulate non-HSL lipase activity in pancreatic islets and this stim-

320 ulatory effect was blocked by the addition of ACBP [25]. Thus, we as-

321 sume that free and ACBP-bound LCAs can affect ATGL activity.

322 Obviously, extensive studies are required to determine the role of

323 ACBPs in lipolysis. In this respect, it is interesting to note that ACBP-

324 deficient mice show a complex metabolic phenotype. These mice go

325 through a crisis with overall weakness at weaning [38] indicating that

326 ACBP is important for metabolic adaption which might also include reg-

327 ulation of lipolysis.

328 The mechanism of LCA-mediated ATGL inhibition appears to be in-

329 dependent of CGI-58, since inactivation of the enzyme was also ob-

330 served in the absence of its co-activator protein and acyl-CoA did not

331 disrupt the protein interaction of ATGL and CGI-58. LCAs were able to

inhibit full-length ATGL and the truncated variant Q289ter lacking the 332 C-terminal domain indicating that they bind to the N-terminal 333 patatin-like phospholipase domain (PNPLA). Furthermore, ATGL was 334 sensitive to LCA inhibition in a heterologous expression system exclud- 335 ing an important contribution of other co-factors. Notably, several 336 PNPLA proteins have been shown to possess acyl-CoA dependent acyl- 337 transferase activity such as adiponutrin [39], GS2 [40], and yeast lipases 338 Tgl3p, Tgl4p, and Tgl5p [41,42]. To our knowledge ATGL does not exhibit 339 this activity, but it is reasonable to assume that ATGL has a yet 340 uncharacterized conserved LCA-binding motif with regulatory function. 341

5. Conclusion 342

LCAs can directly inhibit ATGL in a non-competitive manner. Consid- 343 ering that LCAs also inhibit HSL, LCA-mediated inhibition of lipolytic en- 344 zymes could represent an effective cellular mechanism controlling 345 lipolysis. Inhibition of lipases could be important in adipose and non- 346

adipose tissues, avoid FFA-mediated lipotoxicity, and promote TG accu- 347

mulation when tissues are chronically exposed to high FFA levels as fre- 348

quently observed in obesity. 349

Abbreviations 350

ACBP acyl-CoA binding protein 352

ATGL adipose triglyceride lipase 353

ATGL-ko ATGL-deficient 354

CGI-58 comparative gene identification-58 355

DG diacylglycerol 356

DO diolein 357

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1—1 20

Vmax = 103.3 nmol FFA/h mg Km = 761,8 MM

500 1000 1500 Substrate [mM]

m | 40

^ 30 o

H 20 * 10 0

R2 = 0.9814

10 20 Time [min] • 0 MM 120 MM

ro 400 E

ïî 300 ■

■<3 200

200 400 600 800 1000 Substrate [mM] ■ 0 MM D10MM «20 MM 040 MM

-0.005

0.002 0.004

1/Substrate 0 MM Ü10MM «20mM

358 FFA free fatty acid

359 HSL hormone-sensitive lipase

360 HSL-ko HSL-deficient

361 LCA long-chain acyl-CoA

362 MGL monoglyceride lipase

363 MO monoolein

364 OA oleic acid

365 PNPLA patatin-like phospholipase domain containing protein

366 TG triglyceride

367 WAT white adipose tissue

Fig. 4. Oleoyl-CoA inhibits ATGL in a non-competitive manner. TG hydrolase activity of ATGL was determined in lysates of E. coli overexpressing Strep-tagged ATGL and Strep-tagged CGI-58. (A) Substrate saturation. (B) Time-dependent release of fatty acids in the absence and presence of oleoyl-CoA. (C) Inhibition kinetics raw data. Inhibition kinetics assays where performed in a concentration range of 200 to 800 |M substrate and the indicated concentrations of oleoyl-CoA. (D) Lineweaver-Burk blot of the data shown in (C) indicating a noncompetitive inhibition mechanism. Data are presented as mean ± S.D. from triplicate determinations and representative for two independent experiments.

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369 Acknowledgments

370 This work was supported by the doctoral program Molecular Enzy-

371 mology (Ro. Zi., M. O., Ru. Ze.) and project P22170 (M. O.) funded by

372 the Austrian Science Fund (FWF), and GOLD, Genomics of Lipid-

373 Associated Disorders (Ro. Zi, Ru. Ze.), as part of the Austrian Genome

374 Project GEN-AU funded by the Forschungsförderungsgesellschaft und

375 Bundesministerium für Wissenschaft und Forschung.

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