FEBS Letters 583 (2009) 2527–2534

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Differential effects of chronic, in vivo, PPAR’s stimulation on the myocardialsubcellular redistribution of FAT/CD36 and FABPpm

Agnieszka Kalinowska a, Jan Górski a, Ewa Harasim a, Dorota Harasiuk a, Arend Bonen b, Adrian Chabowski a,*

a Department of Physiology, Medical University of Białystok, 15-089 Białystok, ul. Mickiewicza 2C, Polandb Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1

a r t i c l e i n f o

Article history:Received 27 January 2009Revised 2 July 2009Accepted 7 July 2009Available online 15 July 2009

Edited by Laszlo Nagy

Keywords:Cardiac myocyteFAT/CD36FABPpmPPARLipid

0014-5793/$36.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2009.07.008

* Corresponding author. Fax: +48 085 7485585.E-mail address: adrian@umwb.edu.pl (A. Chabows

a b s t r a c t

This study reveals that the activation of either PPARa (WY 14 643) or PPARb (GW0742) each inducethe translocation of FAT/CD36 from an intracellular pool(s) to the plasma membrane, while PPARbalso induces the subcellular redistribution of FABPpm(Got2) to the plasma membrane. In contrast,activation of PPARc failed to induce the subcellular redistribution of FAT/CD36 and FABPpm. ThesePPARa-, and PPARb-induced changes in the plasmalemmal content of these fatty acid transporterswere associated with the concurrent upregulation of fatty acid triacylglycerol esterification (PPARb)and oxidation (PPARa and PPARb). Observed effects of chronic PPAR stimulation were not related toeither AMPK or ERK1/2 activation.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Long chain fatty acids (LCFAs) are predominant energy sub-strates for the heart [1,2] and thus, the uptake of LCFAs into cardio-myocytes is an essential step in cardiac energy homeostasis. Basedon LCFAs hydrophobic properties, it has been proposed that, theycross the plasma membrane by simple diffusion [3,4]. However, re-cent studies have shown that the majority of LCFAs are taken up bycardiac myocytes via a protein-mediated mechanism [5–7]. So farthree groups of proteins have been identified that contribute tothis process: fatty acid translocase (FAT/CD36), plasma membraneassociated fatty acid binding protein (FABPpm; Got2) and fatty acidtransport proteins 1–6 (FATP1–6) [8–10]. Among them FAT/CD36and FABPpm were found to be the key fatty acid transporters inthe heart as the expression of these proteins in cardiomyocytes ishighly abundant [7,9].

Myocardial expression of LCFA transporters is modulatedthrough both transcriptional and post-transcriptional mecha-nisms [11]. However, recent studies strongly suggest that onlyplasmalemmal abundance of LCFA transporters determines thecardiac myocyte capacity for fatty acid uptake [12–16]. Accord-ingly it was shown that the rates of LCFA transport are upregu-lated, due to the translocation of LCFA transporters from its

chemical Societies. Published by E

ki).

intracellular depot to the plasma membrane. Therefore, by regu-lating the rate of entry of LCFAs into the cells, these LCFAtransport proteins may serve as a key factor contributing to theregulation of lipid metabolism in healthy hearts, whereas in obes-ity and diabetes they may contribute to the development of lipo-toxicity characterized by the excess of lipid accumulation in theheart [2,5,17–19].

Recent studies have demonstrated an important role for theperoxisome proliferator-activated receptors (PPARs) in the tran-scriptional control of genes involved in cardiac fatty acid uptakeand oxidation [20–23]. PPARs are ligand-activated transcriptionfactors of the nuclear hormone receptor superfamily. Three distinctPPAR isoforms termed a, b/d and c have been described in theheart, but PPARa and d are relatively abundantly expressed [24–26]. PPARa activation has been shown to stimulate fatty acids uti-lization in the heart through the transcriptional control of genesresponsible for cardiac fatty acid (FA) uptake, esterification andb-oxidation [21,22,27]. Similarly, PPARb/d stimulation leads toaugmentation of FA transport and oxidation in cardiac myocytes.Importantly these effects were independent of PPARa activation[22,28,29]. The content of PPARc isoform in cardiac muscle cellsis relatively low and its role in cardiac metabolism is still unclear.However, there are indications that PPARc stimulation, in contrastto a and d activation, attenuates fatty acid transmembrane inflowand metabolism while stimulating glucose utilization [22,30].Taken altogether, there is considerable evidence that PPAR

lsevier B.V. All rights reserved.

2528 A. Kalinowska et al. / FEBS Letters 583 (2009) 2527–2534

activation appears to be a key regulator of cardiac substrate metab-olism [23,29–31].

Despite recent studies, little is known regarding the influence ofin vivo PPAR activation on lipid metabolism in the heart muscle,especially their independent effects on LCFAs transport mediatedby fatty acid transport proteins. Therefore, we investigated the ef-fects of chronic, in vivo, PPARs stimulation on the expression (pro-tein and mRNA) and subcellular distribution of the key fatty acidtransporters FAT/CD36 and FABPpm in cardiac myocytes, and onthe rates of fatty acid transport and FA metabolism in the heart.

2. Materials and methods

2.1. Materials

FAT/CD36 and FABPpm were detected as reported previously[32,33] using the (ab 36977, Abcam, UK) and FABPpm antisera (agift from Dr. Calles-Escandon), respectively. The total and phos-phorylated quantities of selected cytosolic proteins were deter-mined with commercially available antibodies total andphospho-ERK1/2 (Thr202/Thr204) and total and phospho-AMPK(Thr172), (Cell Signaling Technology, Beverly, MA). [3H]-palmitatewas purchased from Amersham Life Science (Little Chalfont, UK).Appropriate secondary antibodies were purchased from AbcamUK or Cell Signaling Technology (Beverly, MA). All other chemicalswere obtained from Sigma–Aldrich (St. Louis, MO).

2.2. Methods

Male Wistar rats (250–300 g) were bred on site and maintainedat 20 oC on a reverse light-dark cycle in approved animal holdingfacilities. They had unrestricted access to food (standard chow)and water. The animals were divided into four groups: (i) control(receiving only 0.5% methylcellulose by an oral gavage), or treateddaily for 2 weeks by oral gavage with a selective (ii) PPARa agonist(WY 14 643, 3 mg/kg body weight), (iii) PPARc agonist (pioglitaz-one, 3 mg/kg body weight, Actos, Lilly) or (iv) PPAR b/d agonist(GW0742, 3 mg/kg body weight, Cayman Chemicals). In each groupthe palmitate oxidation and palmitate incorporation into differentlipid pools in the heart was measured. Total (protein and mRNA)FAT/CD36 and FABPpm expressions as well as their plasma mem-branes (PM) and low density microsomes (LDM) content weredetermined. This study was approved by the Ethical Committeefor Animal Experiments at the Medical University of Bialystok.

2.2.1. Palmitate oxidation and incorporation into different lipid poolsTo examine the effects of PPAR’s stimulation on palmitate

metabolism, we used intact hearts that were perfused in theLangendorff perfusion mode. Briefly, after removing hearts fromanaesthetized rats (Vetbutol (50–60 mg/100 g, i.p.), combined withheparin (300 i.u./100 g, i.p.)), the hearts were perfused for a 5 minequilibration period with Krebs Henseleit buffer (KHB). Subse-quently, the hearts were switched to KHB containing 3H-palmitate(100 lmol/l with a corresponding palmitate-to-BSA ratio of 0.3 andcontinuously gassed for 15 min under normoxic (37 oC, 95% O2 and5% CO2) conditions). Thereafter, hearts were removed and immedi-ately freeze-clamped in liquid nitrogen and stored at �80 oC untilanalyzed. Heart lipids were extracted according to van der Vusseet al. [34], with modification as we previously described [35]. Fro-zen heart samples were pulverized in an aluminium mortar with astainless steel pestle, both pre-cooled in liquid nitrogen. The pow-der was transferred into glass tubes containing 2 ml of methanol at�20 �C. Subsequently, 4 ml of chloroform was added. Lipids fromchloroform layer were separated into different fractions by meansof thin-layer chromatography (silica plate 60, 0.25 mm, Merck)

using heptane: isopropyl ether:acetic acid (60:40:3, v/v/v) as a sol-vent. After drying, the plates were sprayed with a 0.2% solution of20,70-dichlorofluorescein in methanol and exposed shortly toammonia vapours. The lipid bands, localized under UV light andidentified according to the standards (Sigma, St. Louis, MO), werescraped off the plates. Finally, samples were dissolved in hexaneand the 3H-palmitate incorporation into different lipid pools wasdetermined. Water-soluble phase of the samples was transferredimmediately into scintillation vials and counted for radioactivity(palmitate oxidation). Oxidation rates of 3H-palmitate were alsodetermined by measuring the release of 3H2O and other water sol-uble intermediates into the buffer at the end of the Langendorffperfusion. The validity of using rates of 3H2O production from3H-palmitate as a measure of palmitate oxidation has been previ-ously described [36]. In brief, 3H2O was separated from 3H-palmi-tate by treating 0.5 ml perfusion buffer with a mixture ofchloroform and 2 M KCL:HCL solution. The aqueous phase wasthen collected and subsequently treated with a mixture ofchloroform, methanol and KCL:HCL (1:1:0.9 vol/vol/vol). Subse-quently, the aqueous phase was removed and counted for radioac-tivity. This technique results in an extraction and separation of3H2O from 3H-palmitate, as has been reported previously [36].The palmitate oxidation was counted as a sum of the 3H2O andwater soluble intermediates present in the heart muscle tissueand in the perfusion buffer at the end of the Langendorf perfusion.Simplificated palmitate uptake was estimated as a sum of the oxi-dation and the esterification.

2.2.2. Subcellular fractionation of intact perfused heartsTo relate changes in the palmitate metabolism with fatty acid

transporters we examined the protein expression and the subcellu-lar distribution of fatty acid transporters. For these purposes con-trol and PPAR’s stimulated hearts were prepared as describedabove, frozen in liquid nitrogen and stored at �80 oC until ana-lyzed. Subcellular fractionation was performed using procedurespreviously described [14–16,37]. Upon thawing, hearts were dicedand incubated for 30 min in a high-salt solution (2 mol/l NaCl,20 mmol/l HEPES pH 7.4, and 5 mmol/l NaN3) at 4 �C as recom-mended by Fuller et al. [38]. Thereafter, the suspension was centri-fuged for 5 min at 1000�g and the pellet homogenized in 6.0 mlTES-buffer using a tightly fitting 10-ml Potter-Elvejhem glasshomogenizer with 10 strokes. The resulting homogenate was cen-trifuged for 5 min at 1000�g, after which the pellet was rehomog-enized in 4.0 ml TES-buffer with 10 strokes and then recombinedwith the 1000�g supernatant. The homogenate was centrifugedfor 10 min at 100�g. The pellet (P1) was resuspended in 300 llTES-buffer and saved. The supernatant was centrifuged for10 min at 5000�g. The pellet (P2) was resuspended in 300 llTES-buffer and saved. The supernatant was centrifuged for20 min at 20 000�g. The pellet (P3) was resuspended in 300 llTES-buffer and saved. The supernatant was centrifuged for30 min at 48 000�g. The pellet (P4) was resuspended in 150 llTES-buffer and saved. The supernatant was centrifuged for65 min at 250 000�g. The pellet (P5) was resuspended in 150 llTES-buffer and saved. Upon analysis of P1–P5 with ouabain-sensi-tive p-nitrophenyl-phosphatase and with EGTA-sensitive Ca2+-ATPase, it has been established that fractions P2 refers as PM-frac-tion and P5 as LDM fraction [14–16,37,39].

2.2.3. Western blottingThe total expression of FAT/CD36 and FABPpm protein was

determined in hearts homogenates yielding crude membranes, asdescribed previously [37,39]. The content of FAT/CD36 and FAB-Ppm was also determined in PM and LDM fractions obtained fromsubfractionated hearts, as described in previous section. Thecytosolic proteins were detected in heart homogenates as de-

A. Kalinowska et al. / FEBS Letters 583 (2009) 2527–2534 2529

scribed previously [37]. Protein content was determined withbicinchoninic acid method with BSA serving as a protein standardSignals obtained by Western blotting were quantified by densitom-etry (BioRad, Poland). Routine Pancou S staining was used to en-sure equal protein loading [14,15,37].

All data are expressed as mean ± S.E.M. Statistical difference be-tween groups was tested with analyses of variance and appropriatepost hoc tests, or with a t-test. Statistical significance was set atP 6 0.05.

2.2.4. FAT/CD36 and FABPpm mRNATotal RNA was isolated from the heart tissue using a commer-

cially available Total RNA Isolation Kit (A&A Biotechnology, Poland)as outlined by the manufacturer. Following RNA purification,DNAse treatment (Ambion, UK) was performed to ensure therewas no genomic DNA contamination. Real time qPCR was per-formed using a Chromo 4 System (Bio-Rad, Poland) by SYBR GreenJumpStart Taq ReadyMix (Sigma, USA). Relative FATCD36 and FAB-Ppm (gene name Got2) RNA levels were calculated using the DDCTmethod and b-actin served as internal control. The following pri-mer sets were used: (1) FAT/CD36: forward: 50-GCAACAACAAGGC-CAGGTAT-30 and reverse: 50-AAGAGCTAGGCAGCATGGAA-30, (2)FABPpm: forward: 50-TCATCCTTTTGTCTCCAGCTTTT-30 and re-verse: 50-CCTATGCCATGCTGACAGGT-30, (3) b-actin: forward: 50-CACACCCGCCACCAGTTC-30 and reverse: 50-GTAGGAGTCCTTCTGACCCATAC-30.

3. Results

3.1. Effects of PPARs on fatty acid transporters

3.1.1. Effects of PPARa stimulation on the total expression andsubcellular redistribution of FAT/CD36 and FABPpm in heart

Chronic in vivo PPARa activation did not change the total myo-cardial protein expression of either FAT/CD36 or FABPpm, although

Fig. 1. Effects of PPARa stimulation on: (1) total protein expression, (2) mRNA and (3) thare shown. Data are based on 10 independent determinations. PM: plasma membranes,

the transcript mRNA was significantly elevated for FAT/CD36 (1.7-fold, P < 0.05). PPARa stimulation induced also the redistribution ofFAT/CD36 from an intracellular pool (LDM: �25%, P < 0.05) to theplasma membranes (PM: +33%, P < 0.05). In contrast, FABPpm sub-cellular distribution remained constant (LDM: �5% and PM: +6%,P > 0.05) (Fig. 1).

3.1.2. Effects of PPARb/d stimulation on the total expression andsubcellular redistribution of FAT/CD36 and FABPpm in heart

Treatment with PPARb/d agonist increased the myocardialexpression of FAT/CD36 at the (1) protein level (+20%, P < 0.05)and (2) mRNA (+2.1-fold, P < 0.05), but had no significant effecton FABPpm expression (protein: +5%, P > 0.05 and mRNA: +1.3-fold, P > 0.05). Surprisingly, PPARb/d stimulation induced the trans-location of both FAT/CD36 and FABPpm to the plasma membranes(PM: +55% and +15%, P < 0.05, respectively) while concomitantlyreducing the content of FAT/CD36 and FABPpm in the intracellularfraction (LDM: �45% and �10%, P < 0.05, respectively) (Fig. 2).

3.1.3. Effects of PPARc stimulation on the total expression andsubcellular redistribution of FAT/CD36 and FABPpm in heart

In contrast to PPARa and PPARd stimulation, chronic PPARcactivation did not alter the myocardial total expressions (proteinand mRNA) of either FAT/CD36 and FABPpm, and had no significantinfluence on their redistribution from an intracellular pool (LDM:P > 0.05, respectively) to the plasma membranes (PM: P > 0.05,respectively) (Fig. 3).

3.2. Effects of PPARs on fatty acid metabolism

3.2.1. Effects of PPARa stimulation on the esterification and oxidationof palmitate in intact, perfused heart

In Langendorff perfused hearts, chronic PPARa stimulation, in-duced significant increase in the incorporation of radiolabelled pal-mitate into only myocardial diacylglycerols (+28%, P < 0.05). There

e subcellular redistribution of FAT/CD36 and FABPpm. Representative Western blotsLDM: low density microsomes. *P < 0.05, PPARa stimulation vs. control.

Fig. 2. Effects of PPARb/d stimulation on: (1) total protein expression, (2) mRNA and (3) the subcellular redistribution of FAT/CD36 and FABPpm. Representative Western blotsare also shown. Data are based on 10 independent determinations. PM: plasma membranes, LDM: low density microsomes. *P < 0.05, PPARb/d stimulation vs. control.

Fig. 3. Effects of PPARc stimulation on: (1) total protein expression, (2) mRNA and (3) the subcellular redistribution of FAT/CD36 and FABPpm. Representative Western blotsare also shown. Data are based on 10 independent determinations. PM: plasma membranes, LDM: low density microsomes. *P < 0.05, PPARc stimulation vs. control.

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was no change in the incorporation of palmitate into the phospho-lipid or triacylglycerol lipid fractions (Fig. 4A). A consistent in-crease in palmitate oxidation after PPARa activation alsooccurred (+40%, P < 0.05) (Fig. 4B).

3.2.2. Effects of PPARb/d stimulation on the esterification and oxidationof palmitate in intact, perfused hearts

Stimulation of PPARb/d did not induce significant changes in theincorporation of radiolabelled palmitate into phospholipids nor

Fig. 4. Effects of PPAR‘s stimulation on: (A) the esterification and (B) oxidation ofpalmitate in intact, Langendorff perfused hearts. Data are based on 10 independentdeterminations for each treatment (mean sem). PL: phospholipids, DG: diacylgly-cerols, TG: triacylglycerols. *P < 0.05, PPAR’s stimulation vs. control.

ig. 5. Correlation between the effects of PPAR’s stimulation on the palmitateptake and plasmalemmal fatty acid transporters expression (as expressed relative

control = 100).

A. Kalinowska et al. / FEBS Letters 583 (2009) 2527–2534 2531

diacylglycerols, however, there was a significant increase in theradioactivity in triacylglycerol myocardial lipid fraction (+34%,P < 0.05) (Fig. 4A). We also found that palmitate oxidation was sig-nificantly greater after PPARb/d activation (+56%, P < 0.05) (Fig. 4B).

3.2.3. Effects of PPARc stimulation on the esterification and oxidationof palmitate in intact, perfused hearts

In contrast to the PPARa and PPARb/d activation, palmitatemyocardial oxidation after PPARc stimulation decreased signifi-cantly (�42%, P < 0.05) (Fig. 4B). Simultaneously, there were no sig-nificant changes in palmitate incorporation into different lipidpools in the intact perfused hearts (Fig. 4A).

3.2.4. Effects of PPAR’s stimulation on the palmitate uptake in intact,perfused hearts

In Langendorff perfused hearts, palmitate uptake was signifi-cantly increased after PPARa and PPARb/d chronic stimulation,whereas there was no change followed PPARc activation. We ob-served that the changes in the palmitate uptake after PPAR’s stim-ulation distinctly correlated with the changes in plasmalemmalcontent of FAT/CD36 (Fig. 5).

3.2.5. Effects of PPAR’s stimulation on cytosolic expression of AMPKand ERK1/2

In vivo, activation of different PPAR‘s had no effect on either thetotal or phoshorylated protein expression of AMPK and ERK1/2(Fig. 6).

4. Discussion

Our present study reveals, that the activation of either PPARa(WY 14 643) or PPARb/d (GW0742) each induce the translocationof FAT/CD36 from an intracellular pool(s) to the plasma membrane,while PPARb/d also induces the subcellular redistribution of FAB-

Futo

Ppm to the plasma membrane. In contrast, activation of PPARcfailed to change the subcellular distribution of FAT/CD36 and FAB-Ppm. These PPARa-, and PPARb/d-induced changes in the plasma-lemmal content of the fatty acid transporters were also associatedwith the concurrent upregulation of fatty acid oxidation. Impor-tantly, the increase in fatty acid oxidation was greater when bothplasmalemmal FAT/CD36 and FABPpm were increased (PPARb/dactivation). These effects were independent of the activation ofeither AMPK or ERK1/2 and were not related to the changes inFFA serum. The administration of either pioglitazone (PPARc) orGW0742 (PPARb/d) decreased serum free fatty acid concentration,but stimulation of PPAR a (WY 14 643) had no effect on serum FFA(Table 1). Previously, rosiglitazone (another PPARc activator) hasbeen found to reduce serum fatty acid concentration in obese Zuc-ker rats [40], but not in obese mice [41]. Similarly, the reduction ofFFA serum levels was observed after the treatment with GW0742[42]. In contrary, treatment with WY 14 643 results in either nochange in circulating fatty acids [43] or reduction in NEFAs (Non-Esterificated Fatty Acids) [44,45]. It is well known that, protein-mediated LCFAs transport depends on plasma concentrations offatty acids and appears to be upregulated when the LCFA milieuis increased by a high fat diet or by fasting [46,47]. Accordingly,neonatal cardiac myocytes exposed to high concentrations of pal-mitate revealed FAT/CD36 mRNA upregulation [47]. Importantly,LCFAs and a variety of related compounds are known ligands forPPARs and observed effects of in these studies likely were relatedto PPARs activation. However, despite the importance of PPARs inthe regulation of cardiac lipid metabolism and energy homeostasis,very few studies examined the influence of PPARs activation on FAtransporters expression [44]. Nonetheless, in our study, serum FFAlevels were not related to the changes in FAT/CD36 and FABPpm(Got2) expressions (protein and mRNA) nor their plasmalemmalcontent (Table 1). This leaves the opportunity to speculate whetherin vivo activation of PPARs directly influences myocardial expres-sion of fatty acids transporters. It has recently been shown thattransgenic mice with cardiac-specific over-expression of PPARa[myosin heavy chain (MHC)-PPAR mice] develop severe cardiomy-opathy associated with marked myocardial lipid accumulation andincreased myocardial expression of FAT/CD36 mRNA [27,48]. Inour present study we show that in vivo PPARa stimulation in-creased transcript FAT/CD36 mRNA content, although did notchange the total protein FAT/CD36 expression. Importantly, PPARaactivation induced also FAT/CD36 translocation to the cardiacmyocyte plasmalemma, with concomitant upregulation in palmi-tate uptake. In contrast to PPARa stimulation, PPARb/d activation

Fig. 6. Effects of PPAR‘s stimulation on the AMPK, ERK1/2 cytosolic expression.

Table 1Summary of the results.

Treatment mRNA Total expression PM expression Uptake Oxydation PL TG DG Serum FFA

PPARa/WY 14 643 CD36 – " FABPpm – NS CD36 – NS FABPpm – NS CD36 – " FABPpm – NS " " NS NS " NSPPARb/d/GW0742 CD36 – " FABPpm – NS CD36 – " FABPpm – NS CD36 – " FABPpm – " " " " " NS " NS ;PPARc/Pioglitazone CD36 – NS FABPpm – NS CD36 – NS FABPpm – NS CD36 – NS FABPpm – NS ; ; NS NS NS ;

NS – not significant." – treatment vs. control.; – treatment vs. control.

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resulted in significant increase in both the total (protein andmRNA) and plasmalemmal FAT/CD36 expression. Moreover, weobserved also FABPpm translocation following PPARb/d stimula-tion. Redistribution of both fatty acid transporters (FAT/CD36 andFABPpm) to the plasma membrane was accompanied by markedincrease in palmitate uptake, which was distinctly greater thanwith PPARa stimulation. These provide strong evidence for PPARaand PPARb/d contribution to the protein-mediated fatty acidstransport in the heart. Previous studies have shown that mice withcardiomyocyte-restricted PPARb/d deletion exhibit impaired lipidmetabolism, progressive myocardial lipid accumulation, cardiachypertrophy and in consequence heart failure [49]. Conversely,chronic treatment with a highly selective PPARb/d agonist(GW610742) in a rat model of congestive heart failure, improvedfat oxidation rate, minimizing adverse manifestations of the dis-ease [29]. The absence of any effect of PPARc on fatty acid trans-porters and metabolism are in accordance with previous studiesin which PPARc activation failed to alter cardiac expression ofFAT/CD36 mRNA and the mRNAs of other metabolic genes[22,47]. Thus, our study and others [22,47] argue against PPARcmajor contribution to the regulation of cardiac lipid metabolism.It is likely that the negligible effects of PPARc activation on fatty

acids transport and oxidation are related to its low abundance inthe heart [22].

Recent evidence has revealed that protein-mediated cardiacfatty acid transport across plasma membranes is regulated byactivation of different intracellular signaling pathways such asPI3K/Akt (insulin) [14,37,39] or AMPK, MAPK (AICAR, contrac-tions) or PKC [15,50,51]. Acute effects of various stimuli inducedthe translocation of only FAT/CD36 (insulin) and at times FABPpm(AMPK activation), from intracellular depots to the plasma mem-brane and in consequence increased the rate of fatty acid uptakeinto cardiomyocytes [14,15,37,39]. Present study shows that pos-sible involvement of either AMPK or ERK1/2 in the redistributionof both FAT/CD36 and FABPpm from intracellular pool to the plas-ma membrane plays rather a minor or permissive role in chronic,in vivo PPARa and PPARb/d activation. It is hard to address in vivoPPAR activation with relation to the intracellular signaling path-ways as too many factors (hormone and/or substrate milieu)may account for the observed effects. Furthermore, it seems likelythat this PPARs effect occurs independently of the activation ofeither AMPK or ERK1/2 as others observed also lack of significantchanges in the expression and activity of AMPK in PPARa defi-cient mice [52].

A. Kalinowska et al. / FEBS Letters 583 (2009) 2527–2534 2533

Our present study (Fig. 5) confirms previous observations show-ing that changes in palmitate uptake are distinctly correlated withthe plasmalemmal content of FAT/CD36. In consideration of oursand other researcher’s findings, FAT/CD36 appears as a majorplayer among fatty acid transporters in the heart [13–15]. How-ever, it is still possible, that FAT/CD36 and FABPpm may collabo-rate in facilitating FA transport across the plasma membrane[5,11,53]. This might be further suggested by PPARb/d activationwhich resulted in far more abundant fatty acid uptake when theplasmalemmal content of both FAT/CD36 and FABPpm was in-creased (Table 1).

The metabolic consequence of PPARa and PPARb/d activation inthe present study was a significant increase in palmitate oxidationin intact, perfused hearts. In contrast, we observed a reduction inpalmitate oxidation after PPARc treatment. Our findings are inaccordance with several previous studies indicating the pivotalrole of PPARa and PPARb/d in fatty acid utilization in cardiac myo-cytes, suggesting that both PPARs share a similar role in cardiaccells regarding lipid metabolism [20,22]. Interestingly, we ob-served a decrease in myocardial FA oxidation followed by PPARc,but we can speculate that PPARc-mediated decrease in FA oxida-tion is rather based on decreased fatty acids plasma concentration.Apart from stimulating fatty acid oxidation, we also observed a sig-nificant increase in palmitate incorporation into diacylglycerolsand triacylglycerols after PPARa and PPARb/d activation, respec-tively. In our opinion, the intensification of fatty acids incorpora-tion into different lipid pools may be simply the result of FAuptake upregulation, as these lipid fractions may serve as tempo-rary fatty acids storage in cardiac myocytes.

5. Conclusions

Our results indicate that PPARa and PPARb/d activation, but notPPARc, can upregulate fatty acid uptake and oxidation in the heartby inducing the translocation of FAT/CD36 (PPARa and PPARb/d)and at times FABPpm (PPARb/d) to the plasma membranes. Impor-tantly, observed effects of chronic PPARs activation were not re-lated to either AMPK or ERK1/2 activation or serum FFA levels.

Acknowledgements

These studies were funded by the Ministry of Science and High-er Education (Research Grant #1-18011) and Medical University ofBiałystok (Grant no. 3-18718). A. Bonen is the Canada ResearchChair in Metabolism and Health. This work was also supported inpart by grants from the Canadian Institutes of Health Research(AB), the Natural Sciences and Engineering Research Council ofCanada (AB), the Heart and Stroke Foundation of Ontario (AB).

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