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Prostaglandins & other Lipid Mediators 85 (2008) 17–25

Bezafibrate decreases growth stimulatory action of the sphingomyelinsignaling pathway in regenerating rat liver

Piotr Zabielski ∗, Marcin Baranowski, Małgorzata Zendzian-Piotrowska,Agnieszka Błachnio-Zabielska, Jan Gorski

Department of Physiology, Medical University of Białystok, Mickiewicza 2c, 15-089 Białystok, Poland

Received 1 April 2007; received in revised form 17 September 2007; accepted 26 September 2007Available online 2 October 2007

bstract

Liver regeneration after partial hepatectomy (PH) is achieved through proliferation of hepatocytes and non-parenchymal cells. The nucleareroxisome proliferator-activated receptor � (PPAR�) is involved in regulation of lipid metabolism and proliferation of hepatic cells. The sphin-omyelin signal transduction pathway is involved in the regulation of the cell cycle in eukaryotic organisms. Sphingosine-1-phosphate (S1P) anderamide (CER) – the intermediates of the pathway – are known to stimulate and to inhibit cellular proliferation. The aim of the present study waso investigate the effect of PPAR� activation by bezafibrate on the sphingomyelin signaling pathway during the first 24 h of liver regeneration afterH in the rat. The content of sphingomyelin, ceramide, sphingosine, sphinganine, sphingosine-1-phosphate and the activity of sphingomyelinasesnd ceramidases were determined at various time points after PH. It has been found that the activity of neutral Mg2+-dependent sphingomyeli-ase (nSMase) increased, whereas the activity of acidic sphingomyelinase (aSMase) decreased in the regenerating liver. Activation of PPAR� by

ezafibrate lower the activity of nSMase and increased the activity of aSMase in the regenerating rat liver. The content of ceramide was highern bezafibrate-treated rats, whereas the content of sphingosine-1-phosphate was markedly lower as compared to the untreated rats. Therefore, it isoncluded that activation of PPAR� by bezafibrate decreases the growth-stimulatory activity of the sphingomyelin pathway in regenerating rat liver.

2007 Elsevier Inc. All rights reserved.

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eywords: Peroxisome proliferator-activated receptors; PPAR�; Bezafibrate; L

. Introduction

The liver possesses the ability to regenerate after partial hep-tectomy (PH) through proliferation of the liver cells. AfterH, cytokines activate hepatocyte pro-survival transcription fac-

ors such as nuclear factor kappa B (NF�B), activation protein(AP-1) and signal transducer and activator of transcription(STAT3) [1] which drive the quiescent hepatocytes from

Abbreviations: PPAR�, peroxisome proliferator-activated receptor alpha;H, partial hepatectomy; TNF�, tumor necrosis factor �; S1P, sphingosine--phosphate; SPH, sphingosine; SPA, sphinganine; CER, ceramide; SM,phingomyelin; Mg2+ nSMase, Mg2+-dependent neutral sphingomyelinase;SMase, acidic sphingomyelinase; aCDase, acidic ceramidase; nCDase, neutraleramidase; SPHK, sphingosine kinase; C-ut, untreated, sham-operated controlroup; C-bf, sham-operated control group treated with bezafibrate.∗ Corresponding author. Tel.: +48 85 7485585; fax: +48 85 7485586.

E-mail addresses: piotrz@amb.edu.pl (P. Zabielski), marcinb@amb.edu.plM. Baranowski), piotrowm@amb.edu.pl (M. Zendzian-Piotrowska),lacha@amb.edu.pl (A. Błachnio-Zabielska), gorski@amb.edu.pl (J. Gorski).

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098-8823/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.prostaglandins.2007.09.001

egeneration; Sphingomyelin pathway; Ceramide; Sphingosine-1-phosphate

he G0 resting state into G1 phase of the cell cycle. Subse-uent stimulation by the growth factors (TGF�-transformingrowth factor alpha, HGF-hepatocyte growth factor and EGF-pidermal growth factor) induces the cell cycle progressionnd finally cell division [2]. The restoration of original mass,ntrinsic structure and function of the liver after 70% partialepatectomy is completed in 7–10 days in the rat [2]. Fattycids and phospholipid emulsions accelerate the process of liveregeneration [3–6]. Proliferating liver cells utilize fatty acidss the main source of energy [7]. Hepatic lipid metabolism isontrolled by ligand-activated transcription factors belongingo the nuclear receptor superfamily. The nuclear peroxisomeroliferator-activated receptor alpha (PPAR�) regulates intra-ellular lipid metabolism through direct transcriptional controlf numerous genes involved in the cellular uptake, intracellu-

ar transport and �-oxidation of fatty acids (reviewed in [8]).ctivation of PPAR� receptor by synthetic ligands (bezafibrate,Y-14.643) up-regulates liver lipid catabolism and decreases

he plasma-free fatty acids, triglyceride and cholesterol level [9].

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8 P. Zabielski et al. / Prostaglandins &

he PPAR� knock-out mice display lower rate of hepatocellularNA synthesis and significantly delayed liver regeneration [10].his further emphasizes the importance of lipid metabolism dur-

ng liver regeneration. Interestingly, the expression of PPAR�eceptor is decreased in rat and murine liver after partial hepatec-omy [11], which could explain the mild liver steatosis observedn the early stages of liver regeneration. Lipids are not onlyhe source of energy and inert constituents of the biological

embranes, but they are also important bioactive compoundsnd play significant role in intra/extracellular signal transduc-ion and regulation of various cellular processes. Sphingomyelinathway of signal transduction influences the fate of eukaryoticells. This pathway is activated by a number of extracellulartimuli such as pro-inflammatory cytokines, ionizing radiation,itamin D3 and growth factors [12]. The major second messen-ers of this pathway are ceramide (CER), sphingosine (SPH) andphingosine-1-phosphate (S1P). CER is released by the hydrol-sis of plasma membrane and lysosomal sphingomyelin (SM)y the action of neutral Mg2+-dependent sphingomyelinasenSMase) and acidic sphingomyelinase (aSMase), respectively13]. Ceramide suppresses the cell growth and induces senes-ence and apoptosis in various cell types [12]. Hydrolysis oferamide by ceramidase (CDase) yields free sphingosine whichnhibits protein kinases and induces apoptosis. Phosphoryla-ion of sphingosine by the enzyme sphingosine kinase (SphK)ields sphingosine-1-phosphate. This bioactive molecule aug-ents cell proliferation and suppresses apoptosis [14,15]. Itas shown in cultured rat hepatocytes that activation of acidic

phingomyelinase by TNF� leads to ceramide generation andubsequent apoptosis. However, activation of neutral ceramidasend the increase in the content of S1P rescued the hepatocytesrom the apoptotic death [16]. We previously showed that par-ial hepatectomy increases the activity of nSMase and neutraleramidase in the remnant liver lobes, which is accompaniedy increase in the contents of SPH and S1P and elevation in1P/CER ratio. This reflects the prevalence of the pro-mitoticignaling of sphingomyelin pathway at early stages of liveregeneration in the rats [17].

As it has already been mentioned, PPAR� plays an impor-ant role in regulation of lipid metabolism in the liver. However,here are no data on an involvement of the PPAR receptors inegulation of the sphingomyelin pathway either in normal oregenerating liver. Therefore, the aim of the present study waso examine the effect of PPAR� synthetic ligand—bezafibraten the functioning of the pathway during the first 24 h of liveregeneration after partial hepatectomy in rat.

. Materials and methods

.1. Animals and study design

The investigation conforms with the “Principle of Labora-ory Animal Care” (NIH Publication no. 85-23, revised 69,

996) and was approved by the Ethical Committee for Ani-al Experiments at the Medical University of Białystok. Maleistar rats (210–230 g of body weight) were fed on a stan-

ard laboratory rodent chow (Agropol, Motycz, Poland), and

2s

1

r Lipid Mediators 85 (2008) 17–25

ad free access to tap water. The rats were divided into twoajor groups: (1) partially hepatectomized-untreated (PH-ut);

2) treated with bezafibrate and then partially hepatectomizedPH-bf). Bezafibrate was administrated at a dose of 7.5 mg/100 gf body weight/day for 7 days by oral gavages, as described pre-iously [18]. The samples of the regenerating liver were takenn 4, 12 and 24 h after the surgery (n = 10 per each time point).

oreover, samples of the normal liver were taken from sham-perated rats (untreated, sham control group; C-ut; n = 10), androm sham-operated rats treated with bezafibrate (sham control-ezafibrate group; C-bf; n = 10). The animals received the lastose of bezafibrate 2 h before the operation. Partial hepatectomyncluded removal of the two-thirds of the liver under pentobar-ital anesthesia, and was performed as described by Higgins andnderson [19]. Rats subjected to partial hepatectomy were kept

n separate cages until sacrifice. The remnant liver was removedt the time points indicated above, frozen in liquid nitrogen andtored until further analysis at −80 ◦C.

.1.1. Sphingomyelinase activityNeutral, Mg2+-dependent sphingomyelinase and acidic sph-

ngomyelinase activity was measured according to Liu andannun [20]. The reaction was carried out in 37 ◦C for0 min in the final volume of 200 �l (0.5 mM [N-methyl-4C]-sphingomyelin, 1221 DPM/nmol, American Radiolabeledhemicals Inc., St. Louis, MO, USA). The amount of hydrolyzed

ubstrate during enzymatic reaction was less than 10% of theotal content. The enzyme activity was calculated by measur-ng the amount of released 14C-phosphocholine (the product ofphingomyelin hydrolysis) with the use of liquid scintillationounter. The activity of sphingomyelinases were expressed inmol of released phosphocholine/mg of protein/h.

.1.2. Ceramidase activityThe activity of acidic and neutral ceramidase was assayed

y measuring the amount of radioactive palmitic acid liber-ted from palmitoyl-1-14C-sphingosine (Moravek Biochemicalsnc., Brea, CA, USA) according to the method by Nikolova-arakashian and Merrill [21]. The incubation was carriedut at 37 ◦C for 1 h with the 50 nmol of [14C]-ceramide2353 DPM/nmol). The released 1-14C-palmitate was extractedith Dole solution (2-propanol/n-heptane/1N NaOH; 40:10:1,/v/v) and n-heptane and quantified by the means of liquid scin-illation counter. The activity of ceramidase was expressed inmol of released palmitate/mg of protein/h.

.1.3. Protein contentThe protein content in samples was measured with the use of

CA-1 kit (Sigma–Aldrich, St. Louis, MO, USA) in a 96-welllate system using an ELISA reader (Multiscan EX, Thermoabsystems Sunnyvale, CA).

.1.4. The content of sphingosine, sphinganine andphingosine-1-phosphate

The content of sphingosine, sphinganine and sphingosine--phosphate was measured according to the method of Min

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t al. [22]. Sphingoid bases were converted to their fluores-ent OPA-derivatives with the use of o-phthalaldehyde (Fluka,uchs, SG, Switzerland) and quantified on the Varian ProStar

iquid chromatograph equipped with Varian OmniSpher 5 C18olumn and fluorescence detector. The mobile phase constitutedf acetonitrile/H2O (9:1, v/v). Quantitative analysis was basedn the standard curves prepared with the use of commerciallyvailable standards of sphingosine, sphinganine and S1P (Avantiolar Lipids, Alabaster, Al, USA). All organic solvents wereurchased from MERCK (Whitehouse Station, NJ, USA).

.1.5. The content of ceramide and sphingomyelinTotal content of ceramide and sphingomyelin was determined

y gas–liquid chromatography (GLC) as described previously23]. Lipids were extracted from pulverized samples by theethod of Folch with the use of ice-cold chloroform/methanol

2:1, v/v) containing 0.01% BHT (2,[6]-di-tert-butylo-p-krezol)wt./v). Sphingomyelins and ceramides were separated fromipid extracts by thin-layer chromatography (TLC) using solventystem comprising of chloroform/methanol/acetic acid/water50:37.5:3.5:2, v/v/v/v) [24] and hexane/diethyl ether/aceticcid (10:90:1, v/v/v) [25], respectively (MERCK Whitehousetation, NJ, USA). Ceramide- and sphingomyelin-fatty acidsere trans-methylated by incubation with 14% methanolic BF3

Sigma–Aldrich, St. Louis, MO, USA) in 100 ◦C for 90 min.atty acid methyl esters were analyzed by gas–liquid chromatog-aphy on Hewlet-Packard 5890 Series II gas chromatographquipped with FID detector. Argon was used as a carrier gas.uantitative and qualitative analysis was based on standard

urves prepared with the use of commercially available fatty acidtandards (Sigma–Aldrich, St. Louis, MO, USA). The amountf sphingomyelin and ceramide is expressed as the sum of indi-idual fatty acid residues in the respective fraction.

.1.6. Statistical analysisResults are shown as mean ± S.D. (n = 10/group). Statisti-

al differences between groups were determined by ANOVA

gm((

ig. 1. The influence of bezafibrate treatment on the content of sphingomyelin (Salues ± S.D.). ��: p < 0.01; ���: p < 0.001 vs. respective values in the control-untntreated group; x: p < 0.05; z: p < 0.001 vs. the respective values in the control-bezafihe PH-untreated groups. The sphingomyelin and the ceramide content in the sampleeramide (CER FA) fraction.

r Lipid Mediators 85 (2008) 17–25 19

or Welch ANOVA in case of non-homogenity of variances)nd subsequent post hoc tests (Tuckey or Dunnet T3 foron-homogenous variances). The significance level was set to≤ 0.05. The values of enzymatic activities and sphingolipid

ontent in liver for both the untreated sham-operated controlnd untreated partially hepatectomized rats were consistent withhose presented in our previous article [17].

. Results

.1. Bezafibrate decreases the content of sphingomyelinnd increases the content of ceramide in rat liver after PHFig. 1)

PPAR� activation by bezafibrate decreased the content ofphingomyelin at 4th, 12th and 24th hour after the operation by1%, 18% and 27% respectively, as compared to the respectivealues in the PH-ut groups (p < 0.001 in each case) (Fig. 1A). Theontent of sphingomyelin in the PH-bf group at 4th and 24th hourfter the surgery was lower by 15% and 11% as compared to theezafibrate-treated control (C-bf; p < 0.05) (Fig. 1A). The con-ent of sphingomyelin in bezafibrate-treated control group wasower by 12% (p < 0.01), as compared to the untreated controlroup (Fig. 1A).

In PH-ut groups the content of ceramide at 4th 12th and4th hour after the operation was higher than the baselinealue by 25% (p < 0.01), 32% (p < 0.001) and 43% (p < 0.001),espectively (Fig. 1B). Bezafibrate significantly increased theontent of ceramide at 12th and 24th hour after PH as com-ared to the respective PH-ut groups (p < 0.001 in each case)Fig. 1B). In the PH-bf groups the content of ceramide at2th and 24th hour after the operation was higher by 35% and

roup (p < 0.001 in each case) (Fig. 1B). Bezafibrate treat-ent increased the content of ceramide in C-bf group by 37%

p < 0.001), as compared to the untreated control group (C-ut)Fig. 1B).

M: A) and ceramide (CER: 1B) in rat liver after partial hepatectomy (meanreated group; b: p < 0.01; c: p < 0.001 vs. the respective values in the control-brate group; ***: p < 0.001 vs. the respective values in the PH-bezafibrate and

s is the sum of individual fatty acids present in the sphingomyelin (SM FA) or

20 P. Zabielski et al. / Prostaglandins & other Lipid Mediators 85 (2008) 17–25

Fig. 2. The influence of bezafibrate treatment on the activity of Mg2+-dependent, neutral sphingomyelinase (Mg2+ nSMase: A) and acidic sphingomyelinase (aSMase:B especv spectr

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) in rat liver after partial hepatectomy (mean values ± S.D.). �: p < 0.05 vs. rs. the respective values in the control-untreated group; z: p < 0.001 vs. the reespective values in the PH-bezafibrate and the PH-untreated groups.

.2. Bezafibrate treatment reduces the activity of nSMasend stimulates the activity of aSMase in rat liver after PHFig. 2)

Partial hepatectomy increased the activity of nSMase in theat liver at 4th, 12th and 24th hour by 29% (p < 0.001), 21%p < 0.01) and 17% (p < 0.05) respectively, as compared to thentreated control group (C-ut; Fig. 2A). At 4th, 12th and 24thour after the surgery nSMase activity in PH-bf groups wasower by 14%, 40% and 59% as compared to the respectiveH-ut groups (p < 0.001 in each case). In PH-bf groups, the activ-

ty of nSMase at 12th and 24th hour after partial hepatectomyecreased by 22% and 49% respectively, as compared with the-bf group (p < 0.001 in both cases).

The activity of aSMase in PH-ut group at 4th hour was lowery 28% as compared to the baseline value (p < 0.001) (Fig. 2B).

t 12th hour after PH the activity of the enzyme returned to theaseline level and at 24th hour after PH it exceeded the controlalue by 16% (p < 0.05). In PH-bf groups the aSMase activity atth and 12th hour after the surgery exceeded the values observed

tpah

ig. 3. The influence of bezafibrate treatment on the activity of neutral ceramidase (nCDmean values ± S.D.). ���: p < 0.001 vs. respective values in the control-untreated group; x: p < 0.05; z: p < 0.001 vs. the respective values in the control-bezafibrate gH-bezafibrate and the PH-untreated groups.

tive values in the control-untreated group; a: p < 0.05; b: p < 0.01; c: p < 0.001ive values in the control-bezafibrate group; *: p < 0.05; ***: p < 0.001 vs. the

n PH-ut groups by 40% (p < 0.001) and 21% (p < 0.05), respec-ively. The aSMase activity at each measured time point in theemnant liver lobes of rats receiving bezafibrate was also signif-cantly higher as compared to the C-bf group (p < 0.001 in eachase) (Fig. 2B). Interestingly, bezafibrate treatment decreasedhe activity of aSMase in C-bf group by 14% (p < 0.05), asompared to the untreated control (C-ut) (Fig. 2B).

.3. Effect of bezafibrate on the activity of nCDase andCDase in regenerating rat liver (Fig. 3)

The two-fold increase in the activity of nCDase in PH-utroup was observed at 12th hour after the operation, as comparedo the C-ut group (p < 0.001) (Fig. 3A). The enzyme’s activityecreased at 24th hour after the surgery, though remained sig-ificantly higher (p < 0.05; by 29%) than in the C-ut group. In

he C-bf group the activity of nCDase was 86% higher as com-ared to the values from C-ut group (p < 0.001) (Fig. 3A). Thectivity of nCDase at 4th hour after PH was almost two timesigher in PH-bf group as compared with respective values in

ase: A) and acidic ceramidase (aCDase: B) in rat liver after partial hepatectomyroup; a: p < 0.05; c: p < 0.001 vs. the respective values in the control-untreatedroup; *: p < 0.05; **: p < 0.01; ***: p < 0.001 vs. the respective values in the

P. Zabielski et al. / Prostaglandins & other Lipid Mediators 85 (2008) 17–25 21

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ig. 4. The influence of bezafibrate treatment on the content of sphingosinealues ± S.D.). ���: p < 0.001 vs. respective values in the control-untreated gr: p < 0.001 vs. the respective values in the control-bezafibrate group; ***: p < 0

H-ut group (p < 0.001). At 24 h after PH the nCDase activity inH-bf group was significantly lower than the respective controlalue (Fig. 3A).

The activity of aCDase in PH-ut groups increased at 4thour after the operation by 39% (p < 0.001) and then returnedo the control value (Fig. 3B). Bezafibrate treatment increasedhe activity of aCDase at 4th and 24th hour after the operationy 21% and 33% as compared to the respective PH-ut groupp < 0.05 in both cases) (Fig. 3B).

.4. Bezafibrate treatment decreases the content ofphingosine (SPH) and sphinganine (SPA) in regeneratingat liver (Fig. 4)

The content of SPH in PH-ut groups at 4th, 12th and 24thour after the operation was higher by 45%, 62% and 62%,espectively, as compared to the control value (p < 0.001 in each

ase) (Fig. 4A). The content of SPH in PH-bf groups was lowery 38% at 4th hour, by 33% at 12th and by 22% at 24th hour afterartial hepatectomy as compared to the respective PH-ut groupp < 0.001 in each case), and higher by 20% (p < 0.05), 45%

s

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ig. 5. The influence of bezafibrate on the content of sphingosine-1-phosphate (S1espective values in the control-untreated group; c: p < 0.001 vs. the respective valuontrol-bezafibrate group; ***: p < 0.001 vs. the respective values in the PH-bezafibr

: A) and sphinganine (SPA: B) in rat liver after partial hepatectomy (mean: p < 0.001 vs. the respective values in the control-untreated group; x: p < 0.05;vs. the respective values in the PH-bezafibrate and the PH-untreated groups.

p < 0.001) and 67% (p < 0.001), respectively, as compared to-bf group. Bezafibrate treatment decreased the content of SPH

n C-bf group by 25%, as compared to the C-ut group (p < 0.001).Partial hepatectomy markedly increased the content of SPA

n the remnant liver lobes. At 4th, 12th and 24th hour the sph-nganine content was two-, three-and almost two times higher,espectively, as compared to the C-ut group (p < 0.001). The con-ent of sphinganine in the remnant lobes of PH-bf rats was alsolevated comparing to the C-bf values (p < 0.001 in each case),owever at 4th and 12th hour after PH it was only about half ofhe respective values in the PH-ut groups (p < 0.001) (Fig. 4B).he SPA level in bezafibrate-treated control group (C-bf) was

ower by 45% (p < 0.001), as compared to the untreated controlC-ut).

.5. Bezafibrate treatment lowers the content of

phingosine-1-phosphate in rat liver after PH (Fig. 5)

The content of sphingosine-1-phosphate in the remnant lobesf PH-ut group at 4th and 12th hour after the operation was

P) in rat liver after partial hepatectomy (mean values ± S.D.). �: p < 0.05 vs.es in the control-untreated group; x: p < 0.05 vs. the respective values in the

ate and the PH-untreated groups.

22 P. Zabielski et al. / Prostaglandins & other Lipid Mediators 85 (2008) 17–25

Table 1The sphingosine-1-phosphate to ceramide ratio (S1P/CER) in rat liver after partial hepatectomy—effect of bezafibrate treatment

Sham 4th hour after PH 12th hour after PH 24th hour after PH

Untreated 1.148 ± 0.135 1.616 ± 0.273c 1.476 ± 0.212b 0.837 ± 0.114bBezafibrate 0.727 ± 0.110��� 1.192 ± 0.185x,*** 0.578 ± 0.107*** 0.609 ± 0.106***

Values expressed in nmol of S1P × nmol−1 of CER × 1000 ± standard deviation (n = 10). ���: p < 0.001 versus respective values in the C-ut group; b: p < 0.01; c:p pectiP

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igher by 74% as compared to the control value (p < 0.001n both cases) (Fig. 5). At 24th hour after PH the content ofhe compound returned to the baseline level. Bezafibrate par-ially prevented the elevation in the content of S1P in remnantobes, at 4th (by 27%, p < 0.001) and at 12th hour (by 47%,< 0.001) as compared to the respective PH-ut group (Fig. 5). In

he bezafibrate-treated control group (C-bf) the S1P content wasower by 12% (p < 0.05), as compared to the untreated controlC-ut).

.6. Effect of bezafibrate on the ratio of the content of S1Po the content of CER (S1P/CER ratio) in regenerating rativer (Table 1)

In the PH-ut group at 4th and 12th hour after the operation the1P/CER ratio exceeded the control value by 41% (p < 0.001)nd 28% (p < 0.01), respectively (Table 1). At 24th hour the ratioas lower by 27% (p < 0.01) than the baseline value. In the PH-f group, the S1P/CER ratio exceeded the control value onlyt 4th hour after the operation (by 61%, p < 0.001). The bezafi-rate reduced the S1P/CER ratio at 4th, 12th and 24th hour afterhe operation by 26%, 61% and 27% respectively (p < 0.001n each case), as compared to respective PH-ut groups. Thishenomenon was also observed between bezafibrate-treated andntreated control groups (reduction by 37%, p < 0.001, as com-ared to the C-ut group).

. Discussion

Bezafibrate treatment strongly affected the sphingomyelinignaling pathway in the liver of both sham-operated andartially hepatectomized rats. PPAR� activation decreasedhe content of sphingomyelin and increased the content oferamide in sham-operated control group (C-bf), as comparedo the untreated control (C-ut). Interestingly, the activity ofoth isoforms of sphingomyelinase (n- and a-SMase) wasecreased in the C-Bf group. This suggest, that either bezafibratencreased the activity of other isoforms of sphingomyeli-ases, or it stimulated de novo pathway of ceramide synthesis.eramide accumulation upon PPAR� activation by WY-14643as observed by Baranowski and others in the heart of rats fedigh-fat diet [26]. This was accompanied by activation of ser-ne palmitoyltransferase (SPT). Authors suggest, that PPAR�

ctivation stimulates de novo pathway of ceramide synthesis ineart. However, bezafibrate treatment decreased the content ofree sphingoid bases (SPA, SFA) in sham control rats as com-ared to the untreated sham control. This suggests that de novo

caat

ve values in the C-bf group; ***: p < 0.001; versus the respective values in the

athway was rather inhibited by PPAR� activation in rat liver,ince both compounds are important intermediates of ceramideynthesis [12]. Therefore, the cause of ceramide elevation underezafibrate treatment in rat liver requires further clarification.

Partial hepatectomy itself increased the activity of nSMase inemnant liver, whereas the treatment with bezafibrate stronglyecreased the activity of the enzyme. It has been shown, thateutral Mg2+-dependent sphingomyelinase is involved in thenduction of crucial pro-survival transcription factors NF�B [27]nd AP-1 [28]. Increased activity of the nSMase is observed initogen-stimulated proliferating vascular smooth muscle cells

29], rat glomerular mesangial cells [30], and primary rat hep-tocytes [16]. In contrast, aSMase is directly involved in thenduction of the programmed cell death in mouse and rat hep-tocytes [16]. Liver cells from aSMase knock-out mice areesistant to TNF�-induced apoptosis [31]. PPAR� activationy bezafibrate could stimulate the pro-apoptotic branch of thephingomyelin pathway in regenerating rat liver by decreasinghe activity of nSMase and stimulating the activity of aSMase.phingomyelin hydrolysis results in the formation of ceramidehich induces apoptosis in a number of cell lines [32]. Bezafi-rate treatment decreased the content of sphingomyelin andncreased the content of ceramide in regenerating rat presum-bly by activation of aSMase. Ceramide can trigger the death ofepatocyte through the activation of caspase-3 [33], disruption ofitochondrial function [34,35] and stimulation of the production

f reactive oxygen species (ROS) [34,36]. Ceramide-inducedncrease in the content of ROS and release of cytochrome-crom mitochondria activate apoptotic proteases and triggers thexecution phase of the programmed cell death [37]. Produc-ion of reactive oxygen species in liver after partial hepatectomys a result of TNF� action [38]. During liver regeneration thencrease in the content of glutathione (GSH) [39] and the activa-ion of superoxide dismutase [38] protects hepatocytes againstytotoxic effects of ROS accumulation. The depletion of cellu-ar glutathione can greatly reduce the liver regenerative potential39]. The activation of PPAR� receptor increases the content ofOS in the liver as a result of acyl-CoA oxidase induction ineroxisomes (which in turn leads to the production of hydrogeneroxide) [40], and by activation of TNF� release from Kupfferells [41]. In human HepG2 cells fenofibrate (a PPAR� agonist)riggers programmed cell death by induction of ROS overpro-uction and subsequent depletion of glutathione [42]. TNF�

an increase both production of reactive oxygen species [43] andctivity of aSMase in rat hepatocytes [31]. The hepatocytes fromSMase knock-out mice (aSMase −/−), are resistant to TNF�-riggered apoptosis under the depletion of cellular glutathione

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ontent [31]. Application of ceramide restored the sensitivityf aSMase knock-outs to apoptosis mediated by TNF� [31]nd FAS ligands [44]. Above-mentioned facts suggest that thencreased content of cellular ceramide can inhibit liver regen-ration in bezafibrate-treated rats, presumably by induction ofxidative stress.

The anti-proliferatory action of ceramide can be lowered bynduction of ceramidase activity [45,46]. Partial hepatectomyriggered the increase in activity of both nCDase and aCDasen liver of PH-ut rats. Concomitantly with the increased activityf both isoforms of the enzyme, we observed the increase inhe content of both SPH and S1P in remnant liver. The increasen the content of S1P under overexpression of nCDase geneas observed in primary rat hepatocytes by Osawa et al. [16].he authors identified S1P as a pro-survival, growth-stimulatory

actor, which protected hepatocytes against cytotoxic effects ofNF�. The anti-apoptotic effect of nCDase activation was due toeramide hydrolysis to sphingosine, but also S1P formation, ashe SPH can be phosphorylated to yield growth-stimulatory S1P.n PH-ut groups partial hepatectomy triggered marked elevationn S1P content, whereas in PH-bf groups similar effect occurrednly at 4th hour after PH. The content of SPH in bezafibrate-reated groups was also significantly lower as compared withppropriate values from PH-ut groups. Interestingly, the nCDasectivity in PH-bf groups was overall higher at 4th hour after theperation as compared with PH-ut group. The increased activityf ceramidases in PH-bf group can be considered as a pro-ective reaction against the elevated ceramide content. Partialepatectomy increases the levels of pro-inflammatory cytokinesuch as TNF�, IL-1 and IL-6 which induce “priming” of theepatocytes [47]. Hepatocytes are usually resistant to the cyto-oxic action of TNF�. In regenerating rat liver the pro-mitoticffects of the TNF� action (such as NF�B activation) prevailver pro-apoptotic (such as increase in reactive oxygen speciesroduction). TNF� activates in hepatocytes a number of pro-urvival enzymes such as phosphatidylinositol 3-kinase (PI3K)48], Akt (PKB) kinase [48,49] and sphingosine kinase (SphK)48,50]. Sphingosine-1-phosphate released by the action of sph-ngosine kinase protects the hepatocytes against both TNF�nd ceramide-mediated apoptosis [48]. The SphK inhibitiony N,N-dimethylsphingosine abolishes the protective effects ofncreased S1P content [6,15,50] or increased nCDase activity16]. The growth inhibitory action of increased CER contents counterweighted by high S1P level [51]. In the bezafibrate-reated groups the S1P/CER ratio was significantly lower thanhe respective values in PH-ut groups. This effect of bezafi-rate treatment can greatly impair survival abilities of hepaticells in regenerating rat liver, as the cytotoxic effects of highNF�/ceramide content cannot be counteracted by increase in

he content of S1P.Partial hepatectomy seems to affect de novo synthesis of

eramide in the remnant liver. In both PH-ut and PH-bf grouphe content of free sphinganine increased. Sphinganine – a

ackbone for complex sphingolipids – is synthesized from-ketosphinganine. The latter is a product of the reactiononducted by serine palmitoyltransferase (SPT) from palmitoyl-oA and l-serine in Golgi apparatus during de novo pathway of

r Lipid Mediators 85 (2008) 17–25 23

phingolipid synthesis. The high activity of SPT was observed inegenerating rat liver and in hepatoma Moris 7777 cell line [52].n our study the content of sphinganine at 12th hour after theperation in PH-ut group was almost five times higher than theaseline. The bezafibrate treatment considerably reduced eleva-ion in the content of sphinganine in the regenerating rat liver.phingolipids are one of the main constituents of cell mem-ranes, thus the rate of sphingolipid synthesis – as reflected byhe cellular content of sphinganine – acts as a limiting factoruring the cellular proliferation. The inhibition of serine palmi-oyltransferase activity decreases the proliferation rate of thehinese hamster ovary cells (line LY-B/cLCB1) [53] and Tcymphocytes (under IL-2 stimulation) [54]. In both cases inclu-ion of the sphingoid backbone’s (sphingosine) in the mediumestored the ability of the cells to proliferate. The promoter site ofhe serine palmitoyltransferase gene does not contain PPAR�-esponsive region, yet the bezafibrate can indirectly influencehe SPT activity. By boosting the rate of fatty acids catabolism55] and by decreasing fatty acids synthesis and esterification56,57] bezafibrate can lower the overall rate of sphingolipidynthesis. The high activity of aSMase, high ceramide and lowphingosine-1-phosphate content in regenerating liver of PH-bfats can be considered as strong growth inhibitory signal. Thencreased content of ceramide can trigger the spiral of death inemnant liver lobes, as the ceramide not only inhibits cellularrowth and triggers apoptosis, but also facilitates the productionf cytotoxic ROS in mitochondria [34,58]. These factors canltimately lead to hepatocyte necrosis [59] and to the inhibitionf liver regeneration in rat.

In summary, we showed that partial hepatectomy increasesro-mitotic activity of the sphingomyelin pathway. It is reflectedy the increased activity of nSMase and ceramidases, increasen the content of S1P and high S1P/CER ratio. Bezafibratereatment decreased above-mentioned factors and increased theontent of ceramide. It is concluded that the activation of PPAR�n regenerating rat liver decreases growth stimulatory activity ofhe sphingomyelin pathway.

cknowledgments

This work was funded by the Polish State Committee forcientific Research grant no. 3P05A 137 25 and the Medicalniversity of Bialystok grant no. 3-18579L.

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