Rat HMGCoA reductase activation in thioacetamide-induced liver ...

Journal of Hepatology 44 (2006) 368–374 www.elsevier.com/locate/jhep

Rat HMGCoA reductase activation in thioacetamide-induced liver injury is related to an increased reactive oxygen species content Valentina Pallottini1,*, Chiara Martini1, Anna M. Bassi2, Paola Romano2, Giorgio Nanni2, Anna Trentalance1 1 Department of Biology, University of Rome ‘Roma Tre’, Viale Marconi 446, 00146—Rome, Italy Department of Experimental Medicine (DIMES), General Pathology Section, University of Genoa, Via L.B. Alberti 2, 16132—Genoa, Italy

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Background/Aims: In thioacetamide-induced liver injury a modification of isoprenoid content and an increase of reactive oxygen species has been described. We have examined how reactive oxygen species influence the 3-hydroxy-3methylglutaryl coenzyme A reductase, the rate limiting enzyme of the isoprenoid biosynthetic pathway, to verify if changes of that enzyme activity are involved in the changed lipid composition of the liver. Methods: In chronic and acute thioacetamide-treated rat liver we measured the reactive oxygen species content, the activation state and KM, the level and degradation rate of the hepatic reductase, its short term regulatory enzymes and the liver lipid profile. Results: In thioacetamide-treated rat liver, the reactive oxygen species content is high and the reductase is fully activated with no modifications in its KM and its short term regulatory enzymes. The reductase level is reduced in chronic thioacetamide treated rats and its degradation rate is altered. Conclusions: The data show a relationship between reactive oxygen species production and altered 3-hydroxy-3methylglutaryl coenzyme A reductase activity. It is suggested that reducing the levels of reactive oxygen species may improve the altered lipid profile found in liver injury. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: HMGCoAR; Thioacetamide; Cholesterol; Liver injury; ROS

1. Introduction The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR, E.C. 1.1.1.34) is an integral membrane protein of the endoplasmic reticulum that represents the key enzyme which catalyses the rate limiting reaction of isoprenoid (i.e. cholesterol, ubiquinone, squalene, dolichol and so on) biosynthesis. It is regulated at multiple levels [1,2]; the long-term regulation is provided by the control of synthesis and

Received 5 April 2005; received in revised form 11 May 2005; accepted 13 June 2005; available online 11 July 2005 * Corresponding author. Tel.: C39 06 5517 6344; fax: C39 06 5517 6321. E-mail address: [email protected] (V. Pallottini).

degradation of the enzyme, while the short-term regulation is mainly assured by phosphorylation or dephosphorylation processes. Thus, HMGCoAR is physiologically present in the cell in an unphosphorylated active form and phosphorylated inactive form. The ratio between active and total enzyme indicates the activation state of the HMGCoAR. Specific kinases and phosphatases, mainly AMP-activated kinase (AMPK) and protein phosphatase 2A (PP2A), are responsible for the active/ inactive form interconversion and represent the enzymes involved in the principal short-term regulation of the HMGCoAR activity [3,4]. The enzyme synthesis is regulated by several hormones, such as thyroid hormones [5] and insulin [6]. The enzyme degradation occurs in the endoplasmic reticulum and is ubiquitin-proteasome system dependent [7]; the binding of some modulators

0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.06.011

V. Pallottini et al. / Journal of Hepatology 44 (2006) 368–374

(mevalonate, 25-hydroxycholesterol, Insig-1) to the sterol sensing domain (SSD) in the membrane-spanning portion of the molecule accelerates HMGCoAR degradation [8], thus the study of the enzyme degradation rate could offer information about the correct assembly of the transmembrane region of the enzyme [9]. In previous works, we reported that in old rats the hepatic HMGCoAR is fully active, its degradation rate is slow, dolichol and cholesterol synthesis are increased, cholesterol content is not changed in the liver but increased in the blood, while dolichol accumulates both in the blood and in the liver [10–13]. The lowered age-related degradation rate of the enzyme could depend on an altered arrangement of its transmembrane domain, and we have suggested that this structural modification could affect the activation state of the enzyme pointing to a strict relationship between the increased reactive oxygen species levels (ROS) and the enzyme deregulation [14]. Beside ageing, studies focused on thioacetamide (TAA)-induced liver injury during acute and chronic treatment highlighted an alteration in dolichol content [15,16], but the mechanism underlying those changes is still under debate and it has never been connected with any change in HMGCoAR activity. TAA is a thiono-sulfur containing compound that is necrogenic [17] and carcinogenic [18]. It is commonly used to induce fulminant hepatic failure [19] and liver cirrhosis [20] in animal models. During the biotransformation of TAA, both flavin-containing mono-oxygenase [21] and cytochrome P450 [22] reduce the oxygen to superoxide anion, which is then catalysed by superoxide dismutase to form hydrogen peroxide (H2O2) [23]. Therefore, biotransformation of TAA produces oxidative damage-associated liver injury. Oxidative damage has previously been implicated because of the glutathione depletion [24], the increase in malondialdehyde [25] and the disappearance of tetraploid hepatocytes [26] observed in the liver following TAA administration. Since nothing is known about the HMGCoAR in TAA-induced liver injury, the aim of this work has been to study the enzyme regulation in this animal model, where both a modified isoprenoid content and oxidative damage are present. For this purpose, in the liver of TAA-treated rats, the activation state, the KM, the shortterm regulatory enzymes, the level and the degradation rate of HMGCoAR were studied, analysing also the lipid profile. The data obtained show that HMGCoAR is fully activated both in acute and in chronic TAA treated animals, while no modifications are detected in its KM and in its short-term regulatory enzymes. The protein level of HMGCoAR and its degradation rate are modified only in the chronic TAA-treated rats, with an increase of the hepatic cholesterol, fatty acid and triglyceride contents.

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2. Materials and methods 2.1. Animals Male Wistar rats (Harlan, San Pietro Natisone, Italy) weighing 230G 10 g were used (7–15 days of acclimatisation). The rats were kept in the animal breeding house, in an environment at constant temperature (228C) and humidity (55%), with a light/dark cycle of 12 h. The Official Italian Regulation N. 116/92 for the care and use of laboratory animals was followed. All rats received a liquid diet (Lieber-De Carli without ethanol, Lab. Piccioni, Milano, Italy). One batch of animals (nZ4) used as controls (C) was put on the liquid diet alone. Another batch (nZ4) (Ch., chronic treated rats) was treated with TAA (Merck, Milano, Italy), at a dose of 300 mg/L of liquid diet, prepared each day, for 1 month. Another batch (Ac., acute treated rats) (nZ4) received 300 mg/kg b.w. of TAA in a single i.p. dose in saline; the animals were sacrificed after 96 h. At the beginning of the experimentation the animals weighed 250G10 g, after 30 days of TAA treatment the weight decreased to 157G4 g, whereas, the control animals after 30 days weighed 357G5 g. The body weight of the acute TAA-treated animals remained unchanged with respect to the control group.

2.2. Measurements of reactive oxygen species ROS were detected on liver homogenates by incubating samples containing 2 mg protein with 10 mM dichlorodihydrofluorescein diacetate (DCF; Molecular Probes, Eugene, OR) for 30 min at 37 8C in the dark, and allowing an additional 60 min equilibration time. We followed the procedure already reported for homogenates and perfused tissues to get a general idea of the extent of oxidative stress [27,28]. Fluorescence was measured under continuous gentle magnetic stirring at 37 8C in a Perkin Elmer LS-50B spectrofluorimeter. Excitation wavelength was set at 498 nm and Emission wavelength was set at 530 nm, using 5 and 10 nm slits, respectively. A Fenton-type reaction initiated by the addition of 10 mM FeSO4 revealed the capability to still produce unstable ROS species.

2.3. HMGCoAR activity assay The assay was carried out with the radioisotopic method, following the production of [14C]-mevalonate from 3-[14C]-hydroxymethylglutaryl coenzyme A (3-[14C]-HMGCoA) (specific activity 57.0 mCi/mmol; Amersham-Pharmacia, Little Chalfont, UK). Five hundred milligrams of liver were homogenised in a phosphate buffer containing 0.1 M sucrose, 0.05 M KCl, 0.04 M KH2PO4, 30 mM EDTA, pH 7.4, in the presence or absence of 50 mM NaF. The activation of the enzyme by lysosomal phosphatases during the preparative procedure can be avoided by the addition of the phosphatase inhibitor NaF. Microsomes were prepared by centrifugation of the homogenates as already reported [29], 100 mg of total microsomal proteins were incubated in the presence of cofactors (20 mM glucose-6-phosphate, 20 mM NADPC sodium salt, 1 IU of glucose-6phosphate dehydrogenase and 5 mM dithiothreitol). The assay, in a final volume of 200 ml, was started by the addition of 10 ml (0.088 mCi/ 11.7 nmol) of 3-[14C]-HMGCoA. The radioactivity of the produced [14C]mevalonate, isolated by chromatography on AG1-X8 ion exchange resin (Bio-Rad Laboratories, Hercules, CA), was counted. An internal standard (3-[3H]-mevalonate, specific activity 24.0 mCi/mmol; Amersham-Pharmacia, Little Chalfont, UK) was added to calculate the recovery. KM determination was obtained by assaying the HMGCoAR activity at substrate concentration ranging from 5 to 164 mM. KM value was calculated from Lineweaver-Burk plot using the GraphPad Prism 4 program.

2.4. Western blot analysis One hundred milligrams of liver tissue were solubilised by sonication in 0.125 M Tris–HCl (pH 7.8) containing 10% SDS, 1 mM phenylmethylsulphonyl fluoride, and boiled for 2 min. Protein concentration was determined by the method of Lowry et al. [30]. A quantity of 20 mg of proteins from solubilised tissue were resolved by 8% SDS-PAGE at 100 V for 60 min, and subsequently transferred electrophoretically onto nitrocellulose for 80 min at 100 V. The nitrocellulose was treated with 3% bovine serum


albumin in 138 mM NaCl, 27 mM KCl, 25 mM Tris–HCl, 0.05% Tween 20 (pH 6.8), probed at room temperature for 1 h with anti-AMPK-P (Cell Signaling Technology, Beverly, MA), and then stripped and probed with anti-AMPK (Cell Signaling Technology). Alternatively the samples were resolved by SDS-PAGE, transferred electrophoretically onto nitrocellulose by following the procedure described above, and then probed with PP2A catalytic subunit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). All the nitrocellulose filters were stripped and probed with the anti-actin antibody (Santa Cruz Biotechnology). The western blotting analysis of HMGCoA reductase was performed on microsomes solubilised in 0.125 M Tris–HCl (pH 7.8) containing 10% SDS, 1 mM phenylmethylsulphonyl fluoride, and boiled for 2 min. Proteins (20 mg) from solubilised microsomes were resolved by 7% SDS-PAGE at 100 V for 60 min. The proteins were subsequently transferred electrophoretically onto nitrocellulose for 80 min at 100 V. The nitrocellulose was treated with 3% bovine serum albumin in 138 mM NaCl, 27 mM KCl, 25 mM Tris–HCl, 0.05% Tween 20 (pH 6.8), probed at 4 8C overnight with HMGCoA reductase antiserum. For all protein detections we used secondary anti-rabbit antibodies (Santa Cruz Biotechnology). Bound antibodies were visualised using enhanced chemoluminescence detection (ECL, Amersham-Pharmacia, Little Chalfont, UK). The arbitrary units were obtained analysing the films with the Fluor-S apparatus using the program Multi-Imager (Bio-Rad Laboratories).

2.5. HMGCoAR degradation in vitro Microsomes, prepared as described above, were suspended in ice-cold 10 mM Tris–HCl (pH 7.4), 150 mM sucrose and then incubated at 37 8C. About 100 mg of proteins were used for each reaction. At the given time, the incubation was terminated by the addition of an equal volume of sample buffer (final concentration 0.125 M Tris–HCl (pH 7.8) containing 10% SDS, 1 mM phenylmethylsulphonyl fluoride), samples were boiled for 2 min, and proteins separated by SDS-PAGE on 7% polyacrylamide gels. Proteins were transferred to nitrocellulose membrane and HMGCoAR was detected by an antiserum. The detection of bound antibody was performed using anti-rabbit IgG and the ECL Western blotting Kit (Amersham-Pharmacia).

2.6. Lipid analysis

*#

400

r=+NaF/-NaF

* *

#

300

150

+NaF -NaF

+NaF -NaF

+NaF -NaF

C

Ch.

Ac.

1

Treatment

Fig. 2. HMGCoAR activity in chronic and acute TAA-treated rat liver. Results are shown for chronic (Ch.) and acute (Ac.) TAA-treated rat liver. HMGCoAR activity was measured following the conversion of [14C]-HMG coenzyme A to [14C]-mevalonate on microsomes prepared in the presence or absence of 50 mM NaF, respectively representing the expressed and the total enzyme activity. Statistical analysis is relative to four different experiments in duplicate. *P!0.001 as from a Student’s t-test with respect to CNaF samples of the control (C); # P!0.005 as from a Student’s t-test with respect to KNaF samples of the control (C). 20!20 (Whatman, Maidston, England), previously activated at 100 8C for 60 min. Samples were developed in petroleum ether/ethyl ether/acetic acid 75:25:1 v/v; bands were visualised with iodine vapour and compared with standards (cholesterol; phospholipids; fatty acids; squalene; triglycerides).

2.7. Statistical analysis Each mean derives from four different experiments performed in duplicate. All the statistical analysis was performed using Student’s t-test.

#

Ch.

(a) C

Ac.

Ch.

Ac.

Treatment HMGCoAR

(b) 25

*# *

It is well known that TAA treatment induces immediate ROS production provoking an oxidative damage [22,31,32].

basal fluorescence fluorescence + FeSO4

#

C

r=0,98

*

0

arbitrary units

arbitrary units

600

0

r=0,95

3. Results

Fifty milligrams of liver tissue was homogenised in chloroform: methanol:H2O 4:2:1 v/v. The mixture was stirred on a vortex mixer for 2 min and then left for 15 min at room temperature. The samples were then centrifuged for 10 min at 600 g. The chloroform fraction was transferred and dried under nitrogen, then the samples were dissolved in 100 ml ethyl ˚ ether and chromatographed on a thin layer chromatography Silica Gel 60 A

200

r=0,49

450 pmol min–1 mg prot–1

370

Treatment

Fig. 1. ROS content in chronic and acute TAA-treated rat liver. ROS were detected by fluorescence measurements incubating the liver homogenates from chronic (Ch.) and acute (Ac.) TAA-treated samples with 10 mM dichlorodihydrofluorescein diacetate for 30 min at 378. Peroxidized or oxidized molecules were assessed by the initiation of a Fenton-type reaction by the addition of FeSO4 (10 mM). For details see the text. Statistical analysis is relative to four different experiments in duplicate. *P!0.001 as from a Student’s t-test with respect to its own control (basal fluorescence); #P!0,001 as from a Student’s t-test with respect to the control (C).

20 *

15 10 5 0

C

Ch.

Ac.

Treatment

Fig. 3. HMGCoA reductase levels. Western blots showing HMGCoAR protein levels in liver microsomes from chronic (Ch.) and acute (Ac.) TAA-treated rats. Proteins from 20 mg solubilised tissue were resolved by SDS-PAGE, and subsequently transferred electrophoretically onto nitrocellulose and probed with anti-HMGCoAR antiserum. Panel (a) shows typical western blotting. Panel (b) shows the densitometric analysis obtained from four different experiments. *P!0.001 as from a Student’s t test with respect to control (C).


Fig. 4. HMGCoAR degradation rate in chronic and acute TAA treated rat liver. Western blots showing HMGCoAR degradation rate from chronic (Ch.) (a) and acute (Ac.) (b) TAA-treated rat liver. The reductase degradation was performed on tissue lysates at 37 8C for the times indicated in a buffer (pH Z7.5) containing Tris-HCl 10 mM and sucrose 75 mM; the HMGCoAR levels were detected by Western blotting. The experiment were performed four times. The panels represent a typical western blotting.

In this work, we first controlled the ROS content in the liver of acute and chronic TAA-treated rats; an increase of the basal ROS content in both groups was detected (Fig. 1). Moreover, the stimulation of a Fenton-type reaction revealed the presence of increased levels of peroxidated molecules confirming an altered red ox state of the cells. The HMGCoAR activity is regulated by phosphorylation-dephosphorylation mechanisms, with the unphosphorylated form representing the active enzyme and the

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phosphorylated form the inactive. The expressed and total enzyme activities were measured in microsomes prepared in the presence or absence of NaF, respectively. The expressed (CNaF) activity increased in the liver of both acute and chronic TAA-treated rats, with respect to the control, and the total activity of the chronic treated rats (KNaF) showed a slight but statistically significant decrease (Fig. 2). The CNaF/KNaF ratio (r), which reflects the activation state of the enzyme in vivo, increased from 0.49 in the control animals to 0.95–0.98 after acute or chronic treatment with TAA (Fig. 2), while the enzyme KM remained unchanged (0.10–0.12 mM for all three batches of animals), indicating that in a damaged liver HMGCoAR regulation may be impaired, but its specific activity is not affected. The amount of the HMGCoAR protein detectable in the TAA-treated liver showed a significant decrease only in the chronic treated animal as assessed by western blot analysis (Fig. 3a,b), in agreement with the lower total HMGCoAR activity detected in the same animals (Fig. 2). In attempt to understand the mechanism underlying the reduced HMGCoAR protein level, the enzyme degradation rate was analysed. Fig. 4a,b shows that the chronic TAAtreatment reduces the enzyme degradation rate, while the acute TAA-treatment does not affect it. To understand whether the HMGCoAR impaired phosphorylation could depend on modification of its specific regulatory enzymes, the levels of PP2A and AMP-activated kinase, responsible, respectively for HMGCoAR dephosphorylation and phosphorylation, were measured. Neither

Fig. 5. AMPK activation state and PP2A catalytic subunit level in chronic and acute TAA treated rat liver. Panel (a) represents the AMPK activation state, panel (b) the PP2A cathalytic subunit levels of chronic and acute TAA-treated rat liver. Proteins from 20 mg solubilised tissue were resolved by SDS-PAGE. The proteins were subsequently transferred electrophoretically onto nitrocellulose and probed with anti-AMPK-P antibody, then stripped and probed with anti-AMPK antibody. The same samples were resolved by SDS-PAGE, transferred electrophoretically onto nitrocellulose and probed with anti PP2A catalytic subunit. For details see the text. The panels represent typical western blots and the densitometric analysis was obtained from four different experiments.

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Fig. 6. Lipid profile of chronic and acute TAA-treated rat liver. The panel a represents the scanner image of a typical TLC plate on which were chromatographed lipid extracts of chronic and acute TAAtreated rat livers. Line 1 Control (C); line 2 Chronic (Ch.); line 3 Acute (Ac.); line 4 cholesterol standard (Chol); line 5 Phospholipids standard (PL); line 6 Fatty Acids standard (FA); line 7 Squalene standard (Sq); line 8 Triglycerides standard (Tr). The panel b shows a table containing the densitometric analysis, expressed in arbitrary units, of the spots from four different experiments. *P!0.001 as from a Student’s t-test with respect to control (C).

the level of the PP2A catalytic subunit nor the activation state of AMPK showed any difference between TAA-treated rats and the controls (Fig. 5a,b). These data suggest that the full activation of the reductase is not caused by changes in the short-term regulatory enzymes. To evaluate the effects of the TAA-induced total HMGCoAR activation state, we analysed the hepatic lipid profile. Fig. 6a,b shows an increase of cholesterol, fatty acids and triglycerides in both chronic and acute treatment, while surprisingly the squalene rises only in acute TAAtreated rats.

4. Discussion TAA is experimentally used for inducing fulminant hepatic failure [19], necrosis [17], carcinogenesis [18] and liver cirrhosis [20] in animal models, and TAA biotransformation determines an abnormal ROS production that produces oxidative damage associated with liver injury. Previously, we have demonstrated that a modified dolichol content was secondary to full HMGCoAR

activation [11], and that an higher HMGCoAR activation state was strictly related to enhanced ROS content [14]. In this view, it was interesting to investigate the HMGCoAR behaviour in TAA-treated rats, where the enhanced ROS content was detectable just after 96 h TAA-treatment. We have confirmed an oxidation state in our animal model that is paralleled by an increased HMGCoAR activation state, with no modifications of both its KM and its short term regulatory enzymes. This picture is common to other systems in which high ROS contents are detectable, such as ageing or young rats subjected to antioxidant depleteddiets [14]. In all these experimental models, where the ROS content is high, the HMGCoAR results fully activated. During ageing hepatic cholesterol content remains unchanged while dolichol accumulates and hypercholesterolemia is observed [11]. In acute TAA-treated rat hepatocytes, a slight dolichol decrease is detectable after 96 hours treatment, probably due to its peroxidation by ROS; however this decline is rapidly reverted [15]. In chronic TAA-treated rats, the dolichol content increases in hepatocytes [33] and the present experiments show that the hepatic cholesterol, fatty acid and triglyceride contents arise too. Thus, the full HMGCoAR activation seems to be the reason for the increased content of its final products. Interestingly, the liver accumulation of cholesterol is observable only in a pathological experimental model (i.e. TAA-treated animals) and not in a physiological one (i.e. ageing), suggesting that in the hepatic injury other factors involved in the maintenance of the cholesterol homeostasis could also be affected, such as proteins involved in cholesterol efflux or in the conversion to bile acids [34, 35]. Furthermore it is attractive the TAA-induced squalene accumulation in the liver of acute treated animals; it might be due to a rapid sterol-induced inhibition of epoxidase [36, 37] whose physiological activity could be restored in chronic TAA-treated rats. The relationship between HMGCoAR activation and ROS is not yet clear. The HMGCoAR degradation rate is strictly dependent on a correct arrangement of the SSD in the membrane and can be lowered when this portion of the enzyme is modified and unable to bind some specific factors, for example Insig-1 [8]. So a change of the degradation rate could represent a good signal of the changed structure of the membrane spanning domain of the enzyme [9]. During ageing, a connection between the presence of a low degradation rate and full activation of the reductase has been suggested. In particular, it was hypothesised that alteration of the membrane spanning portion, caused by the increased ROS content, might affect the structure (twisting or misfolding) of the aminoacid sequence encompassing the phosphorylation-dephosphorylation site (i.e. Ser 871) [13]. Actually the use of different experimental models has shown that the increased ROS content is always able to increase the catalytic activity [14], but not the rate of the enzyme degradation; therefore, it is evident that a modified degradation rate is not always


related to the HMGCoAR full activation. The question that arises is: what is the cause of the different degradation rates? We do not know the answer, but we cannot exclude that the metabolic changes characterizing physiological or pathological situations could be involved; for example during ageing [38] or in chronic TAA-treated liver [32] the enzyme degradation could be a part of a regulatory network that also includes a reduced protein biosynthesis. In conclusion, our data clearly support a direct correlation between ROS production and altered HMGCoAR activity, even if the definition of the underlying mechanism requires further investigation. It is interesting to consider that the altered lipid profile found in liver injury might be avoided by the control of HMGCoAR regulation, that in turn depends on ROS content.

Acknowledgements The authors wish to thank Dr Alessandra Cona (Dept. of Biology, University of Rome ‘Roma Tre’) for the KM result calculation and interpretation. This work was supported by grants from the University of Rome ‘Roma Tre’ 2003 and MIUR Cofin 2003 to A.T.

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