- Email: [email protected]
Adverse effect of fenofibrate on branched-chain α-ketoacid dehydrogenase complex in rat's liver
Toxicology 266 (2009) 1–5
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Adverse effect of fenofibrate on branched-chain ␣-ketoacid dehydrogenase complex in rat’s liver Malgorzata Knapik-Czajka ∗ , Anna Gozdzialska, Jerzy Jaskiewicz Jagiellonian University Medical College, Faculty of Pharmacy, Department of Analytical Biochemistry, Medyczna 9 St., 30-688 Krakow, Poland
a r t i c l e
i n f o
Article history: Received 12 August 2009 Received in revised form 30 September 2009 Accepted 1 October 2009 Available online 9 October 2009 Keywords: Fenofibrate Branched-chain ␣-ketoacid dehydrogenase complex (BCKDH) BCKDH kinase (BDK) Leucine
a b s t r a c t Branched-chain ␣-ketoacid dehydrogense complex (BCKDH) is a regulatory enzyme of valine, isoleucine and leucine catabolism. Its activity is mainly regulated by covalent modification achieved by a specific BCKDH kinase (BDK) and phosphatase (BDP). The goal of our study was to investigate the effect of increasing doses of fenofibrate on BDK and BCKDH activities in rat’s liver. For 14 days fenofibrate was administrated to Wistar male rats (fed chow containing 8% protein) at one of the daily doses: 5, 10, 20 and 50 mg/kg. Control group was given only vehicle (0.3% methylcellulose). BDK activity as well as actual BCKDH activity and total BCKDH activity were assayed spectrophotometrically and BDK protein amount was determined by Western blotting. In rats administered fenofibrate BDK activity decreased by 61%, 64%, 66% and 89% (p < 0.0001). Changes in BDK protein expression did not correspond with changes in BDK activity. BCKDH complex actual activity was 3.7 ± 0.3, 4.1 ± 0.1, 4.6 ± 0.3 and 4.0 ± 0.3 fold higher (p < 0.0001) and BCKDH total activity 1.3 ± 0.1, 1.3 ± 0.1, 1.5 ± 0.1 and 1.3 ± 0.1 fold higher comparing to control group (p < 0.001). BCKDH activity state (percentage of active, dephosphorylated form) increased 2.8 ± 0.2, 3.1 ± 0.1, 3.2 ± 0.1 and 3.0 ± 0.1 fold (p < 0.0001). In addition, fenofibrate prevented body weight gain starting from the dose of 10 mg/kg/day and induced hepatomegaly in a dose-dependent manner. It can be concluded that fenofibrate under condition of protein restriction starting from the lowest dose inhibits BDK activity at the posttranslational level and increases BCKDH activity state. It is conceivable that fenofibrate decreases of branched-chain amino acids (BCAA) levels by stimulation of their catabolism. Since leucine plays an important role in up-regulation of protein anabolism in muscles, the reduced level of this amino acid may be one of the factors involved in pathomechanism of myopathy observed during treatment with fenofibrate. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Branched-chain amino acids (BCAA) leucine, isoleucine and valine are essential nutrients that are indispensable for body protein synthesis. In addition, they exert different regulatory functions including stimulation of protein synthesis, supplying amino groups for the synthesis of the neurotransmitter glutamate and stimulation of hormones secretion (Hutson et al., 2001; Ijichi et al., 2003; Li et al., 2003). It was shown that excess of BCAA and their metabolites can be toxic especially to central nervous system, as manifested in patients with maple syrup urine disease (Mitsubuchi et al., 2005). Because BCAA cannot be stored in any form other than protein they must be catabolised efficiently when their intake exceeds the body’s requirements and conserved when dietary intake of these amino acids is insufficient. Mitochondrial branched-chain ␣-ketoacid dehydrogense complex (BCKDH) is a rate limiting enzyme of BCAA catabolic
∗ Corresponding author. Tel.: +48 12 620 56 60; fax: +48 12 657 02 62. E-mail address: [email protected] (M. Knapik-Czajka). 0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.10.002
pathway. It catalyses irreversible oxidative decarboxylation of branched-chain ␣-ketoacids: ␣-ketoisovalerate, ␣-keto-methylvalerate and ␣-ketoisocaproate, transamination products of valine, isoleucine and leucine, respectively. BCKDH is composed of multiple copies of three catalytic subunits: branched-chain dehydrogenase (E1 component, heterodimer ␣2 2 , EC 1.2.4.4), dihydrolipoamide acyltransferase (E2 component, lack of EC number) with a covalently attached lipoic acid cofactor, and dihydrolipoamide dehydrogenase (E3 component EC 1.8.1.4) (Wynn et al., 1996). As a key enzyme in BCAA catabolism that allows to balance the body’s needs for BCAA with the supply of BCAA from the diet BCKDH activity is precisely regulated. It is generally believed that the reversible phosphorylation of E1␣ subunit (mainly at Ser 262) is a major mechanism involved in BCKDH activity regulation (Harris et al., 1986, 2004; Zhao et al., 1994a). There are two regulatory enzymes associated with BCKDH that participate in covalent modification of the complex: a specific kinase (BDK-BCKDH kinase EC 2.7.1.115) and a specific phosphatase (BDP EC 3.1.3.52) (Harris et al., 1982, 1997). Phosphorylation of E1␣ subunit by BDK leads to inactivation of BCKDH while dephosphorylation catalyzed by
2
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
BDP reactivates the complex. Current catalytic activity of BCKDH described as BCKDH activity state depends on the proportion of BCKDH occurring in active dephosphorylated form and is determined by the relative activities of BDK and BDP. Although BDP has been purified from bovine kidney it has not been fully proven to be the phosphatase responsible for regulation of BCKDH activity (Damuni et al., 1984). Recently Lu et al. (2009) have presented data demonstrating that protein phosphatase 2Cm is the endogenous phosphatase required for nutrient-mediated regulation of BCKDH activity. Regulation of BDK activity and its role in BCAA metabolism has been extensively studied, mostly in rat’s tissues (Shimomura et al., 1990; Popov et al., 1995; Harris et al., 1997). BDK activity is regulated in a short-term manner by different physiological compounds, the most specific allosteric inhibitor being ␣-ketoisocaproate, the transamination product of leucine (Han et al., 1987). In addition, long-term adaptive changes in the level of BDK protein associated with BCKDH as well as the amount of catalytic subunits of the complex occur in response to various factors and conditions including different protein content in the diet. It was shown that in rats fed low-protein chow liver BDK activity and protein amount increase and BCKDH activity and subunit expression decrease (Espinal et al., 1986; Doering and Danner, 2000; Zhao et al., 1994b). In this condition BCKDH activity state and consequently BCAA degradation decreases (Harris et al., 1996). Such a precise physiological regulation system prevents BCAA from degradation and allows for their conservation for protein synthesis. Restriction of protein intake by feeding rats chow containing less than optimal amount of protein provides an experimental model for studying the effect of different factors on BDK and BCKDH activities. It was demonstrated that BDK and BCKDH activities and protein expression are also regulated by different xenobiotics including fibrates such as clofibrate, bezafibrate and fenofibrate (Honda et al., 1991; Paul et al., 1996; Knapik-Czajka et al., 2003). Fibrates constitute a group of hypolipidemic agents that are commonly used in the treatment of hypertriglyceridemia and combined hyperlipidemia, being particularly effective in lowering of the plasma triglyceride concentration (Balfour et al., 1990; Chapman, 2003). At the molecular level the effect of fibrates is mediated by a specific group of transcription factors named peroxisome proliferator activated receptors (PPARs) (Schoonjans et al., 1996). PPARs activation results in change of transcription of target genes encoding for different proteins. Regulation of transcription of genes for proteins involved in lipid metabolism is the most widely studied effect of fibrates as PPARs agonists. It was shown that fibrates or their active metabolites administration to rats fed low-protein chow cause an opposite effect to a physiological regulation of BDK and BCKDH activities (Kobayashi et al., 2002; Knapik-Czajka et al., 2002; Knapik-Czajka and Jaskiewicz, 2003). Fenofibrate, similarly to bezfibrate and clofibrate reduce liver BDK activity, mRNA and protein expression as well as increases the amount of protein subunits of the complex leading to upregulation of BCKDH activity. All of the data concerning fenofibrate effect on BCKDH were obtained with addition of fenofibrate to low-protein chow at a single dose of 0.5%. The current study was undertaken to investigate the in vivo effect of increasing doses of fenofibrate on BDK and BCKDH activities in the liver of rats fed low-protein chow. 2. Materials and methods
ICN Biomedicals (USA). Materials used for Western blotting analysis including nitrocellulose membrane (Hybond-ECL), ECL Plus Western Blotting Detection Reagents and Hyperfilm ECL were obtained from Amersham Biosciences (UK). 2.2. Animals and experimental treatment The study protocol was approved by Jagiellonian University Ethic Committee. Male Wistar rats (from inbred strain) weighting 248.8 ± 8.2 g were purchased from the breeding facilities of the Jagiellonian University Faculty of Pharmacy. They were housed in groups of four in plastic cages at an artificial 12-h light/dark cycle and at constant room temperature (21–23 ◦ C). Rats were fed a low-protein chow (8% protein content) ad libitum and allowed free access to water. After acclimatization to low-protein chow and the oral administration of 0.3% dimethylcellulose rats were randomized into five treatment groups (n = 4). Fenofibrate was administrated by gastric gavage to consecutive groups at one of the daily doses: 5, 10, 20 and 50 mg/kg body weight. Control group was given only vehicle (0.3% methylcellulose). During the treatment body weight and food intake were monitored daily. After 14 days rats were sacrificed and liver tissues were excised, weighted and immediately freezeclamped with aluminum tongs precooled in liquid nitrogen and then stored at −80 ◦ C until analysis. 2.3. Determination of enzymes activities Extracts of tissues for the BCKDH and BDK were prepared as described previously (Goodwin et al., 1988). BCKDH was concentrated from whole tissue extracts prior to assay by precipitation with 9% polyethylene glycol. BCKDH complex activity was determined spectrophotometrically at 30 ◦ C by measuring the rate of NADH generation from NAD+ in the presence of the saturating concentration of ␣-ketoisovaleric acid, a substrate for BCKDH complex (Cary 100 spectrophotometer – Varian). One unit of BCKDH complex activity is defined as the amount of enzyme that catalyzed the formation of 1 mol of NADH/min. The activity of the BCKDH complex occurring in partly dephosphorylated form (actual activity) or completely dephosphorylated form (total activity) were determined before and after incubation with a broadspecificity phosphatase, respectively. The broad-specificity phosphatase necessary to complete dephosphorylation of BCKDH complex was isolated from bovine heart according to the procedure described by Goodwin et al. (1988). Percentage of BCKDH complex in active dephosphorylated state was calculated from values of actual and total activities of BCKDH complex and expressed as activity state of BCKDH complex. For the determination of BDK activity BCKDH complex was completely activated by dephosphorylation with broad-specificity phosphatase and then precipitated again with 9% polyethylene glycol. BDK activity was assayed by determination of the first-order rate constant for ATP-dependent inactivation of BCKDH complex activity in the presence of the saturating concentration of ␣-ketoisovaleric acid. 2.4. Western blotting analysis of BDK protein expression SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis BDK protein expression were performed, using antiBDK rabbit antibodies as described previously (Paul et al., 1996). Protein concentrations were determined by biuret method. Samples (60 g protein/lane) were run on 10% polyacrylamide gel and electrotransferred to a nitrocellulose (ECL) membrane. Secondary antibodies were goat anti-rabbit antibodies (Abcam, UK). Immunoreactive bands on the membranes were detected with a chemiluminescence system and quantified by densitometry (Bio-Rad Quantity One Image analysis software). Relative expression level of BDK protein in each fenofibrate-treated group was presented using arbitrary units (% of control group). 2.5. Statistics Data are presented as mean ± SEM. Differences between groups were determined with one-way analysis of variance (ANOVA), followed, when appropriate, by Bonferroni post hoc test, with acceptable significance levels set at p < 0.05. The initial- and post-treatment body weight values were compared by Student’s t-test for paired samples.
3. Results Considering that fenofibrate vehicle was 0.3% methylcellulose, if not specifically indicated, we referred here to methylcellulose administrated animals as to “control group or control rats”. 3.1. Fenofibrate effect on body and liver weight and food intake
2.1. Materials Fenofibrate (2-[4[(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid]-1methylethyl ester) and all reagents necessary for assay of enzyme activities as well as basic reagents for Western blotting were purchased from Sigma–Aldrich Chemical Company (Germany). Low-protein (8% protein) chow was bought in
The effect of multiple doses of fenofibrate on body weight, food intake and liver to body weight ratio is presented in Table 1. After 14 days of the experiment the body weight of both control and rats administered 5 mg/kg/day fenofibrate increased very
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
3
Table 1 Effect of increasing doses of fenofibrate on body weight, liver weight and daily food intake. Fenofibrate dose (mg/kg/day) 0 Initial body weight (g) Final body weight (g) Liver weight (% of body weight) Food intake (g per group/day)
246 267 3.1 63
5 ± ± ± ±
8 13# 0.3 9
246 267 3.8 56
10 ± ± ± ±
7 9# 0.4 10
251 268 4.5 55
20 ± ± ± ±
11 23 0.2* 10
252 259 4.8 50
50 ± ± ± ±
2 11 0.4* 8*
249 229 5.3 37
± ± ± ±
12 17* 0.3* 12*
Data are presented as mean ± SEM (n = 4). Liver weight is expressed as a percent of body weight. Food intake is presented as daily food intake in g per tested group. * Significantly different from control group (p < 0.05). # Significantly different from initial body weight for respective group (p < 0.05).
slightly but significantly by 9% as comparing to initial body weight (p < 0.05). Final body weight of the groups receiving 10 and 20 mg/kg/day were 7% and 3% higher than at the beginning of the experiment (p > 0.05). On the contrary, the final body weight of animals treated with the highest dose decreased by 8% (p > 0.05). Final body weights of rats treated with 5 and 10 mg/kg/day fenofibrate dose were almost identical as control group. Animals given 20 and 50 mg/kg/day had lower final body weight by 3% and 14%, respectively. There was a significant difference between the group administered with 50 mg/kg/day of fenofibrate when compared to control as well as compared to rats treated with 5 and 10 mg/kg/day (p < 0.05). Liver weight (expressed as a percent of body weight) was dosedependently higher by 21%, 44%, 53% and 68% in fenofibrate-treated rats than in control group (p < 0.0001). The difference was statistically significant starting from the dose of 10 mg/kg/day. Daily food intake decreased in a dose-related manner and was significantly lower in rats given 20 and 50 mg/kg/day fenofibrate than in control group (p < 0.0001).
3.3. Fenofibrate effect on BDK protein expression Changes in BDK protein expression did not correspond with changes in BDK activity. There was not any significant difference in BDK protein expression between fenofibrate-treated groups and control rats (100%; 104%, 96% and 99% vs. 100% for control, respectively) (Fig. 2). 3.4. Fenofibrate effect on BCKDH activity In all fenofibrate-treated rats both actual and total BCKDH complex activities increased significantly in response to graded levels of administered drug starting from the lowest dose (Fig. 3). BCKDH actual activity was 3.7 ± 0.3, 4.1 ± 0.1, 4.6 ± 0.3 and 4.0 ± 0.3 fold higher than in control group (p < 0.0001). BCKDH total activity increased by 1.3 ± 0.1, 1.3 ± 0.1, 1.5 ± 0.1 and 1.3 ± 0.1 fold in animals receiving 5, 10, 20 or 50 mg/kg/day fenofibrate, respectively (p < 0.001). Consequently, BCKDH activity state (the percentage of active, dephosphorylated form) rats was 2.8 ± 0.2,
3.2. Fenofibrate effect on BDK activity Comparing to control group liver BDK activity decreased significantly in a dose-dependent manner by 61%, 64%, 66% and 89% in rats receiving 5, 10, 20 and 50 mg/kg/day of fenofibrate, respectively (p < 0.0001) (Fig. 1).
Fig. 1. Effect of increasing doses of fenofibrate on BDK activity in rat’s liver. Rats were fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Data are presented as mean ± SEM (n = 4). BDK activity is expressed as the first-order rate constant of ATP-dependent inactivation of BCKDH complex. *Significantly different from control group (p < 0.0001).
Fig. 2. Western blot analysis of BDK amounts in liver of rats fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Upper panel: representative immunoblot. Lower panel: bar graphs showing the expression level (arbitrary units-% of control) of BDK protein in each treatment group. Data are presented as mean ± SEM (n = 4).
4
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
Fig. 3. Effect of increasing doses of fenofibrate on BCKDH actual activity (A), total activity (B) and activity state (C) in rat’s liver. Rats were fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Data are presented as mean ± SEM (n = 4). BCKDH total activity was determined after complete dephosphorylation of BCKDH complex with broad-specificity phosphatase. Activity state means percentage of BCKDH complex in active dephosphorylated state. *Significantly different from low-protein group (p < 0.0001). **Significantly different from low-protein group (p < 0.001).
3.1 ± 0.1, 3.2 ± 0.1 and 3.0 ± 0.1 fold higher than in control group (p < 0.0001). There were no statistical differences in actual and total BCKDH activity as well as BCKDH activity state among particular groups treated with different doses of fenofibrate (Fig. 3). 4. Discussion To our knowledge the dose-dependent effect of fenofibrate on BDK and BCKDH activities has not been studied yet. Results of the present study demonstrated that fenofibrate disturbed physiological regulation of BDK. Administration of increasing doses of fenofibrate to rats fed low-protein chow have a dose-related reduction in liver BDK activity. However changes in BDK protein expression did not correspond with changes in BDK activity. Therefore, it is possible that fenofibrate at tested doses affects BDK activity on posttranslational level. It was established that clofibrate and its active metabolite, clofibric acid regulate BDK activity by different mechanisms including direct inhibitory effect on BDK activity (Kobayashi et al., 2002). Clofibric acid is structurally related to ␣-ketoisocaproate, a physiological allosteric inhibitor of BDK (Paxton and Harris, 1984). Because fenofibrate similarly to clofibrate belongs to the group of fibric acid derivatives it is conceivable that in used doses it may inhibit BDK activity directly without changing the amount of BDK protein. In condition of reduced BDK activity BDP activity prevails and higher proportion of BCKDH E1␣ subunit occurs in an active dephosphorylated form what results in higher activity state of BCKDH. Significant increase in liver BCKDH activity state was observed with 5 mg/kg/day dose, and subsequent higher doses did not further increase significantly BCKDH activity state. Probably inhibition of BDK activity by the lowest tested dose of fenofibrate was sufficient to allow almost complete dephosphorylation of BCKDH E1␣ subunit by BDK. So further decrease in BDK activity caused by increasing doses of fenofibrate did not result in any significant increase in BCKDH activity state. Since one of the adverse effects of treatment with fenofibrate is myopathy (Hodel, 2002) it is worth considering the relationship between fenofibrate effect on BCKDH complex and myopathy. It is possible that that fenofibrate-induced up-regulation of BCKDH complex activity leads to reduction in BCAA levels which in turn may affect BCAA-regulated processes. BCAA that account for about
35% amino acids in muscle proteins play a special role in muscle metabolism (Shimomura et al., 2001). Leucine is thought to promote muscle protein anabolism not only by an increase in substrate availability. Leucine stimulates muscle protein synthesis by different mechanism including induction of both the activity and synthesis of proteins involved in mRNA translation (May and Buse, 1989; Crozier et al., 2005; Anthony et al., 2001). In addition, leucine inhibits muscle protein degradation (Louard et al., 1990; Nair et al., 1992). It was shown that BCAA catabolism enhanced by clofibrate treatment has significant influence on the leucineinduced activation of protein translation (Ishiguro et al., 2006). It cannot be excluded that fenofibrate disturbs regulation of muscle protein synthesis and/or degradation. Precisely balanced protein turnover is critical to maintain metabolic homeostasis. Even a small decrease in protein synthesis or acceleration of protein degradation can result in disturbances of physiological balance between these two crucial processes. Therefore, it is conceivable that the reduced levels of leucine may be one of the factors involved in pathomechanism of the myopathy observed during treatment with fenofibrate. Numerous studies that used animals fed different chows demonstrated a negative effect of fenofibrate on body weight, adiposity and food intake (Chaput et al., 2000; Mancini et al., 2001; Larsen et al., 2003). In the current study it was shown that fenofibrate effect on body weight was also present in rats fed low-protein chow. Fenofibrate starting from the dose of 10 mg/kg/day prevented body weight gain while daily food intake was reduced in rats administered with 20 and 50 mg/kg/day. This observation suggests that not only insufficient dietary food intake but also fenofibrateinduced metabolic changes could be the cause of the lack of body weight gain. Fenofibrate was shown to reduce food intake and increase resting energy expenditure by induction of the fatty acids catabolism and uncoupling proteins synthesis in rat liver (Mancini et al., 2001; Park et al., 2007). It is well documented that administration of fibrates to rodents results in numerous hepatic alterations, including hepatomegaly, an increase in the number and size of peroxisomes, and an increase in the expression of genes encoding peroxisomal, mitochondrial, and microsomal fatty acid-metabolizing enzymes (Balfour et al., 1990; Reddy and Chu, 1996). We observed a dose-related increase in the liver weight to body weight ratio in rats treated with fenofibrate starting from the dose of 10 mg/kg/day. Results of the present study demonstrated that fenofibrate at used doses induced hepatomegaly in dose-dependent manner in rats submitted to dietary protein restriction.
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
5. Conclusions It can be concluded that under condition of dietary protein restriction fenofibrate starting from the lowest dose inhibits BDK activity at posttranslational level and increases BCKDH complex activity state. That opposite to physiological regulation effect of fenofibrate may result in up-regulation of BCAA catabolism and decrease in their levels. Leucine, valine and isoleucine are essential amino acids which must be continuously available for body protein synthesis. In addition, leucine promotes muscle protein anabolism by different mechanisms. It cannot be excluded that the reduction of BCAA levels induced by up-regulation of their degradation is one of the factors involved in the pathomechanism of myopathy, one of most serious adverse effect observed during treatment with fenofibrate. Conflict of interest None declared. Acknowledgements This study was supported by State Committee for Scientific Research (KBN) grant number 3 P05F 032-25 (2003–2007). Authors would like to thank Jagoda Drag, MSc and Katarzyna Wadowska for their excellent technical support and Zuzanna Mikosza, MSc for her help in preparation of the manuscript. References Anthony, J.C., Anthony, T.G., Kimball, S.R., Jefferson, L.S., 2001. Signaling pathways involved in translational control of protein synthesis in skeletal muscle by leucine. J. Nutr. 131, 856–860. Balfour, J.A., McTavish, D., Heel, C., 1990. Fenofibrate. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in dyslipidaemia. Drugs 40, 260–290. Chapman, M.J., 2003. Fibrates in 2003: therapeutic action in atherogenic dyslipidaemia and future perspectives. Atherosclerosis 171, 1–13. Chaput, E., Saladin, R., Silvestre, M., Edgan, A.D., 2000. Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochem. Biophys. Res. Commun. 271, 445–450. Crozier, S.J., Kimball, S.R., Emmert, S.W., Anthony, J.C., Jefferson, L.S., 2005. Oral leucine administration stimulates protein synthesis in rat skeletal muscle. J. Nutr. 135, 376–382. Damuni, Z., Merryfield, M.L., Humphreysn, J.S., Reed, L., 1984. Purification and properties of branched-chain ( ketoacid dehydrogenase phosphatase from bovine kidney. Proc. Natl. Acad. Sci. U.S.A. 81, 4335–4338. Doering, C.B., Danner, D.J., 2000. Amino acid deprivation induces translation of branched-chain (-keto acid dehydrogenase kinase. Am. J. Physiol. Cell. Physiol. 279, 1587–1594. Espinal, R., Beggs, M., Patel, H., Randle, P.J., 1986. Effects of low-protein diet and starvation on the activity of branched-chain 2-oxo acid dehydrogenase kinase in rat liver and heart. Biochem. J. 237, 285–288. Goodwin, G.W., Zhang, B., Patton, R., Harris, R.A., 1988. Determination of activity and activity state of branched-chain (-ketoacid dehydrogenase in rat tissues. Methods Enzymol. 166, 189–201. Han, A.C., Goodwin, G.W., Paxton, R., Harris, R.A., 1987. Activation of branched chain (-ketoacid dehydrogenase in isolated hepatocytes by branched chain (ketoacids. Arch. Biochem. Biophys. 258, 85–94. Harris, R.A., Paxton, R., Parker, R.A., 1982. Activation of the branched-chain ␣ketoacid dehydrogenase complex by a broad specificity protein phosphatase. Biochem. Biophys. Res. Commun. 107, 1497–1503. Harris, R.A., Paxton, R., Powell, S.M., Goodwin, G.W., Kuntz, M.J., Han, A.C., 1986. Regulation of branched-chain ␣-ketoacid dehydrogenase complex by covalent modification. Adv. Enzyme Regul. 250, 219–237. Harris, R.A., Goodwin, G.W., Paxton, R., Dexter, P., Powell, S., Zhang, B., Han, A., Shimomura, Y., Gibson, R., 1996. Nutritional and hormonal regulation of the activity state of hepatic branched-chain ␣-ketoacid dehydrogenase complex. Ann. N. Y. Acad. Sci. 804, 307–313. Harris, R.A., Hawes, J., Popov, K.M., Zhao, Y., Shimomura, Y., Sato, J., Jaskiewicz, J., Hurley, T.D., 1997. Studies on the regulation of the mitochondrial ␣-ketoacid dehydrogenase complexes and their kinases. Adv. Enzyme Rev. 37, 271–293. Harris, R.A., Joshi, M., Jeoung, N.H., 2004. Mechanism responsible for regulation of branched-chain amino acid catabolism. Biochem. Bipohys. Res. Commun. 313, 391–396. Hodel, C., 2002. Myopathy and rhabdomyolysis with lipid-lowering drugs. Toxicol. Lett. 128, 159–168.
5
Honda, K., Ono, K., Mori, T., Kochi, H., 1991. Both induction and activation of the branched-chain 2-oxo acid dehydrogenase complex in primary-cultured rat hepatocytes by clofibrate. J. Biochem. 109, 822–827. Hutson, S.M., Lieth, E., LaNoue, K.F., 2001. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J. Nutr. 131, 846–850. Ijichi, C., Matsumura, T., Tsuji, T., Eto, Y., 2003. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem. Biophys. Res. Commun. 303, 59–64. Ishiguro, H., Katano, Y., Nakano, I., Ishigami, M., Hayashi, K., Honda, T., Goto, H., Bajotto, G., Maeda, K., Shimomura, Y., 2006. Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver. Life Sci. 79, 737–743. Knapik-Czajka, M., Cyganek, I., Tyszka-Czochara, M., Francik, R., Jaskiewicz, J., 2002. Effect of fibrates on the branched-chain ␣-ketoacid dehydrogenase complex in rat liver. Acta Pol. Toxicol. 10, 191–198. Knapik-Czajka, M., Tyszka-Czochara, M., Jaskiewicz, J., 2003. Effect of fibrates on the branched chain ␣-ketoacid dehydroganase complex activity. Acta Toxicol. 11, 59–65. Knapik-Czajka, M., Jaskiewicz, J., 2003. The influence of fibrates on the branchedchain ␣-ketoacid dehydrogenase complex expression in rat’s liver. Acta Toxicol. 11, 67–73. Kobayashi, R., Murakami, T., Obayashi, M., Nakai, N., Jaskiewicz, J., Fujiwara, Y., Shimomura, Y., Harris, R.A., 2002. Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms. Arch. Biochem. Biophys. 407, 31–240. Larsen, P.J., Jensen, P.B., Sorensen, R.V., Larsen, L.K., Vrang, N., Wulff, E.M., Wassermann, K., 2003. Differential influences of peroxisome proliferator-activated receptors ␥ and ␣ on food intake and energy homeostasis. Diabetes 52, 2249–2259. Li, C., Najafi, H., Daikhin, Y., Nissim, I.B., Collins, H.W., Yudkoff, M., Matschinsky, F.M., Stanley, C.A., 2003. Regulation of leucine-stimulated insulin secretion and glutamine metabolism in isolated rat islets. J. Biol. Chem. 278, 2853–2858. Louard, R.J., Barrett, E.J., Gelfand, R.A., 1990. Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man. Clin. Sci. 79, 457–466. Lu, G., Sun, H., She, P., Youn, J.Y., Warburton, S., Ping, P., Vondriska, T.M., Cai, H., Lynch, C.J., Wang, Y., 2009. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells. J. Clin. Invest. 119, 1678–1687. Mancini, F.P., Lanni, A., Sabatino, L., Moreno, M., Giannino, A., Contaldo, F., Colantuoni, V., Goglia, F., 2001. Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats. FEBS Lett. 491, 154–158. May, M.E., Buse, M.G., 1989. Effects of branched-chain amino acids on protein turnover. Diabetes Metab. Rev. 5, 227–245. Mitsubuchi, H., Owada, M., Endo, F., 2005. Markers associated with inborn errors of metabolism of branched-chain amino acids and their relevance to upper levels of intake in healthy people: an implication from clinical and molecular investigations on maple syrup urine disease. J. Nutr. 135, 1565–1570. Nair, K.S., Schwartz, R.G., Welle, S., 1992. Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. Am. J. Physiol. Endocrinol. Metab. 263, 928–934. Park, M.-K., Lee, H.-J., Hong, S.-H., Choi, S.-S., Yoo, Y.H., Lee, K.I., Kim, D.K., 2007. The increase in hepatic uncoupling by fenofibrate contributes to a decrease in adipose tissue in obese rats. J. Kor. Med. Sci. 22, 235–241. Paul, H.S., Liu, W.Q., Adibi, S.A., 1996. Alternation in gene expression of branchedchain ketoacid dehydrogenase kinase but not in gene expression of its substrate in the liver of clofibrated rat. Biochem. J. 317, 411–417. Paxton, R., Harris, R.A., 1984. Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain ␣-ketoacid dehydrogenase kinase in vitro and in perfused rat heart. Arch. Biochem. Biophys. 231, 58–66. Popov, K.M., Zhao, Y., Shimomura, Y., Jaskiewicz, J., Kedishvilli, N.Y., Irwin, J., Goodwin, G.W., Harris, R.A., 1995. Dietary control and tissue expression of branched-chain ␣-ketoacid dehydrogenase complex kinase. Arch. Biochem. Biophys. 316, 148–154. Reddy, J., Chu, R., 1996. Peroxisome proliferator-induced pleiotropic responses: pursuit of phenomenon. Ann N. Y. Acad. Sci. 804, 176–201. Schoonjans, K., Staels, B., Auwerx, J., 1996. Role of peroxisome proliferator-activated receptor [PPAR] in mediating the effects of fibrates and fatty acid on gene expression. J. Lipid Res. 37, 907–925. Shimomura, Y., Nanaumi, N., Suzuki, M., Popov, K.M., Harris, R.A., 1990. Purification and partial characterization of branched-chain ␣-ketoacid dehydrogenase kinase from rat liver and rat heart. Arch. Biochem. Biophys. 283, 293–299. Shimomura, Y., Obayashi, M., Murakami, T., Harris, R., 2001. Regulation of branchedchain amino acid catabolism: nutritional and hormonal regulation of activity and expression of the branched-chain ␣-keto acid dehydrogenase kinase. Curr. Opin. Clin. Nutr. Metab. Care 4, 419–423. Wynn, R.M., Davie, J.R., Chuang, D.T., 1996. Structure, function and assembly of mammalian branched-chain ␣-keto acid dehydrogenase complex. In: Patel, M.S., Roche, T.E., Harris, R.A. (Eds.), Alpha-keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel, Switzerland, pp. 101–118. Zhao, Y., Hawes, J., Popov, K.M., Jaskiewicz, J., Shimomura, Y., Crabb, D.W., Harris, R.A., 1994a. Site directed-mutagenesis of phosphorylation sites of the branchedchain ␣-ketoacid dehydrogenase complex. J. Biol. Chem. 269, 18583–18587. Zhao, Y., Hawes, J., Popov, K.M., Jaskiewicz, J., Shimomura, Y., Kedishvilli, N.Y., Jaskiewicz, J., Kuntz, M.J., Kain, J., Hang, B., Harris, R.A., 1994b. Effect of dietary protein on the liver content and subunit composition of the branched-chain ␣-ketoacid dehydrogenase complex. Arch. Biochem. Biophys. 308, 446–453.
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Adverse effect of fenofibrate on branched-chain ␣-ketoacid dehydrogenase complex in rat’s liver Malgorzata Knapik-Czajka ∗ , Anna Gozdzialska, Jerzy Jaskiewicz Jagiellonian University Medical College, Faculty of Pharmacy, Department of Analytical Biochemistry, Medyczna 9 St., 30-688 Krakow, Poland
a r t i c l e
i n f o
Article history: Received 12 August 2009 Received in revised form 30 September 2009 Accepted 1 October 2009 Available online 9 October 2009 Keywords: Fenofibrate Branched-chain ␣-ketoacid dehydrogenase complex (BCKDH) BCKDH kinase (BDK) Leucine
a b s t r a c t Branched-chain ␣-ketoacid dehydrogense complex (BCKDH) is a regulatory enzyme of valine, isoleucine and leucine catabolism. Its activity is mainly regulated by covalent modification achieved by a specific BCKDH kinase (BDK) and phosphatase (BDP). The goal of our study was to investigate the effect of increasing doses of fenofibrate on BDK and BCKDH activities in rat’s liver. For 14 days fenofibrate was administrated to Wistar male rats (fed chow containing 8% protein) at one of the daily doses: 5, 10, 20 and 50 mg/kg. Control group was given only vehicle (0.3% methylcellulose). BDK activity as well as actual BCKDH activity and total BCKDH activity were assayed spectrophotometrically and BDK protein amount was determined by Western blotting. In rats administered fenofibrate BDK activity decreased by 61%, 64%, 66% and 89% (p < 0.0001). Changes in BDK protein expression did not correspond with changes in BDK activity. BCKDH complex actual activity was 3.7 ± 0.3, 4.1 ± 0.1, 4.6 ± 0.3 and 4.0 ± 0.3 fold higher (p < 0.0001) and BCKDH total activity 1.3 ± 0.1, 1.3 ± 0.1, 1.5 ± 0.1 and 1.3 ± 0.1 fold higher comparing to control group (p < 0.001). BCKDH activity state (percentage of active, dephosphorylated form) increased 2.8 ± 0.2, 3.1 ± 0.1, 3.2 ± 0.1 and 3.0 ± 0.1 fold (p < 0.0001). In addition, fenofibrate prevented body weight gain starting from the dose of 10 mg/kg/day and induced hepatomegaly in a dose-dependent manner. It can be concluded that fenofibrate under condition of protein restriction starting from the lowest dose inhibits BDK activity at the posttranslational level and increases BCKDH activity state. It is conceivable that fenofibrate decreases of branched-chain amino acids (BCAA) levels by stimulation of their catabolism. Since leucine plays an important role in up-regulation of protein anabolism in muscles, the reduced level of this amino acid may be one of the factors involved in pathomechanism of myopathy observed during treatment with fenofibrate. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Branched-chain amino acids (BCAA) leucine, isoleucine and valine are essential nutrients that are indispensable for body protein synthesis. In addition, they exert different regulatory functions including stimulation of protein synthesis, supplying amino groups for the synthesis of the neurotransmitter glutamate and stimulation of hormones secretion (Hutson et al., 2001; Ijichi et al., 2003; Li et al., 2003). It was shown that excess of BCAA and their metabolites can be toxic especially to central nervous system, as manifested in patients with maple syrup urine disease (Mitsubuchi et al., 2005). Because BCAA cannot be stored in any form other than protein they must be catabolised efficiently when their intake exceeds the body’s requirements and conserved when dietary intake of these amino acids is insufficient. Mitochondrial branched-chain ␣-ketoacid dehydrogense complex (BCKDH) is a rate limiting enzyme of BCAA catabolic
∗ Corresponding author. Tel.: +48 12 620 56 60; fax: +48 12 657 02 62. E-mail address: [email protected] (M. Knapik-Czajka). 0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.10.002
pathway. It catalyses irreversible oxidative decarboxylation of branched-chain ␣-ketoacids: ␣-ketoisovalerate, ␣-keto-methylvalerate and ␣-ketoisocaproate, transamination products of valine, isoleucine and leucine, respectively. BCKDH is composed of multiple copies of three catalytic subunits: branched-chain dehydrogenase (E1 component, heterodimer ␣2 2 , EC 1.2.4.4), dihydrolipoamide acyltransferase (E2 component, lack of EC number) with a covalently attached lipoic acid cofactor, and dihydrolipoamide dehydrogenase (E3 component EC 1.8.1.4) (Wynn et al., 1996). As a key enzyme in BCAA catabolism that allows to balance the body’s needs for BCAA with the supply of BCAA from the diet BCKDH activity is precisely regulated. It is generally believed that the reversible phosphorylation of E1␣ subunit (mainly at Ser 262) is a major mechanism involved in BCKDH activity regulation (Harris et al., 1986, 2004; Zhao et al., 1994a). There are two regulatory enzymes associated with BCKDH that participate in covalent modification of the complex: a specific kinase (BDK-BCKDH kinase EC 2.7.1.115) and a specific phosphatase (BDP EC 3.1.3.52) (Harris et al., 1982, 1997). Phosphorylation of E1␣ subunit by BDK leads to inactivation of BCKDH while dephosphorylation catalyzed by
2
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
BDP reactivates the complex. Current catalytic activity of BCKDH described as BCKDH activity state depends on the proportion of BCKDH occurring in active dephosphorylated form and is determined by the relative activities of BDK and BDP. Although BDP has been purified from bovine kidney it has not been fully proven to be the phosphatase responsible for regulation of BCKDH activity (Damuni et al., 1984). Recently Lu et al. (2009) have presented data demonstrating that protein phosphatase 2Cm is the endogenous phosphatase required for nutrient-mediated regulation of BCKDH activity. Regulation of BDK activity and its role in BCAA metabolism has been extensively studied, mostly in rat’s tissues (Shimomura et al., 1990; Popov et al., 1995; Harris et al., 1997). BDK activity is regulated in a short-term manner by different physiological compounds, the most specific allosteric inhibitor being ␣-ketoisocaproate, the transamination product of leucine (Han et al., 1987). In addition, long-term adaptive changes in the level of BDK protein associated with BCKDH as well as the amount of catalytic subunits of the complex occur in response to various factors and conditions including different protein content in the diet. It was shown that in rats fed low-protein chow liver BDK activity and protein amount increase and BCKDH activity and subunit expression decrease (Espinal et al., 1986; Doering and Danner, 2000; Zhao et al., 1994b). In this condition BCKDH activity state and consequently BCAA degradation decreases (Harris et al., 1996). Such a precise physiological regulation system prevents BCAA from degradation and allows for their conservation for protein synthesis. Restriction of protein intake by feeding rats chow containing less than optimal amount of protein provides an experimental model for studying the effect of different factors on BDK and BCKDH activities. It was demonstrated that BDK and BCKDH activities and protein expression are also regulated by different xenobiotics including fibrates such as clofibrate, bezafibrate and fenofibrate (Honda et al., 1991; Paul et al., 1996; Knapik-Czajka et al., 2003). Fibrates constitute a group of hypolipidemic agents that are commonly used in the treatment of hypertriglyceridemia and combined hyperlipidemia, being particularly effective in lowering of the plasma triglyceride concentration (Balfour et al., 1990; Chapman, 2003). At the molecular level the effect of fibrates is mediated by a specific group of transcription factors named peroxisome proliferator activated receptors (PPARs) (Schoonjans et al., 1996). PPARs activation results in change of transcription of target genes encoding for different proteins. Regulation of transcription of genes for proteins involved in lipid metabolism is the most widely studied effect of fibrates as PPARs agonists. It was shown that fibrates or their active metabolites administration to rats fed low-protein chow cause an opposite effect to a physiological regulation of BDK and BCKDH activities (Kobayashi et al., 2002; Knapik-Czajka et al., 2002; Knapik-Czajka and Jaskiewicz, 2003). Fenofibrate, similarly to bezfibrate and clofibrate reduce liver BDK activity, mRNA and protein expression as well as increases the amount of protein subunits of the complex leading to upregulation of BCKDH activity. All of the data concerning fenofibrate effect on BCKDH were obtained with addition of fenofibrate to low-protein chow at a single dose of 0.5%. The current study was undertaken to investigate the in vivo effect of increasing doses of fenofibrate on BDK and BCKDH activities in the liver of rats fed low-protein chow. 2. Materials and methods
ICN Biomedicals (USA). Materials used for Western blotting analysis including nitrocellulose membrane (Hybond-ECL), ECL Plus Western Blotting Detection Reagents and Hyperfilm ECL were obtained from Amersham Biosciences (UK). 2.2. Animals and experimental treatment The study protocol was approved by Jagiellonian University Ethic Committee. Male Wistar rats (from inbred strain) weighting 248.8 ± 8.2 g were purchased from the breeding facilities of the Jagiellonian University Faculty of Pharmacy. They were housed in groups of four in plastic cages at an artificial 12-h light/dark cycle and at constant room temperature (21–23 ◦ C). Rats were fed a low-protein chow (8% protein content) ad libitum and allowed free access to water. After acclimatization to low-protein chow and the oral administration of 0.3% dimethylcellulose rats were randomized into five treatment groups (n = 4). Fenofibrate was administrated by gastric gavage to consecutive groups at one of the daily doses: 5, 10, 20 and 50 mg/kg body weight. Control group was given only vehicle (0.3% methylcellulose). During the treatment body weight and food intake were monitored daily. After 14 days rats were sacrificed and liver tissues were excised, weighted and immediately freezeclamped with aluminum tongs precooled in liquid nitrogen and then stored at −80 ◦ C until analysis. 2.3. Determination of enzymes activities Extracts of tissues for the BCKDH and BDK were prepared as described previously (Goodwin et al., 1988). BCKDH was concentrated from whole tissue extracts prior to assay by precipitation with 9% polyethylene glycol. BCKDH complex activity was determined spectrophotometrically at 30 ◦ C by measuring the rate of NADH generation from NAD+ in the presence of the saturating concentration of ␣-ketoisovaleric acid, a substrate for BCKDH complex (Cary 100 spectrophotometer – Varian). One unit of BCKDH complex activity is defined as the amount of enzyme that catalyzed the formation of 1 mol of NADH/min. The activity of the BCKDH complex occurring in partly dephosphorylated form (actual activity) or completely dephosphorylated form (total activity) were determined before and after incubation with a broadspecificity phosphatase, respectively. The broad-specificity phosphatase necessary to complete dephosphorylation of BCKDH complex was isolated from bovine heart according to the procedure described by Goodwin et al. (1988). Percentage of BCKDH complex in active dephosphorylated state was calculated from values of actual and total activities of BCKDH complex and expressed as activity state of BCKDH complex. For the determination of BDK activity BCKDH complex was completely activated by dephosphorylation with broad-specificity phosphatase and then precipitated again with 9% polyethylene glycol. BDK activity was assayed by determination of the first-order rate constant for ATP-dependent inactivation of BCKDH complex activity in the presence of the saturating concentration of ␣-ketoisovaleric acid. 2.4. Western blotting analysis of BDK protein expression SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis BDK protein expression were performed, using antiBDK rabbit antibodies as described previously (Paul et al., 1996). Protein concentrations were determined by biuret method. Samples (60 g protein/lane) were run on 10% polyacrylamide gel and electrotransferred to a nitrocellulose (ECL) membrane. Secondary antibodies were goat anti-rabbit antibodies (Abcam, UK). Immunoreactive bands on the membranes were detected with a chemiluminescence system and quantified by densitometry (Bio-Rad Quantity One Image analysis software). Relative expression level of BDK protein in each fenofibrate-treated group was presented using arbitrary units (% of control group). 2.5. Statistics Data are presented as mean ± SEM. Differences between groups were determined with one-way analysis of variance (ANOVA), followed, when appropriate, by Bonferroni post hoc test, with acceptable significance levels set at p < 0.05. The initial- and post-treatment body weight values were compared by Student’s t-test for paired samples.
3. Results Considering that fenofibrate vehicle was 0.3% methylcellulose, if not specifically indicated, we referred here to methylcellulose administrated animals as to “control group or control rats”. 3.1. Fenofibrate effect on body and liver weight and food intake
2.1. Materials Fenofibrate (2-[4[(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid]-1methylethyl ester) and all reagents necessary for assay of enzyme activities as well as basic reagents for Western blotting were purchased from Sigma–Aldrich Chemical Company (Germany). Low-protein (8% protein) chow was bought in
The effect of multiple doses of fenofibrate on body weight, food intake and liver to body weight ratio is presented in Table 1. After 14 days of the experiment the body weight of both control and rats administered 5 mg/kg/day fenofibrate increased very
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
3
Table 1 Effect of increasing doses of fenofibrate on body weight, liver weight and daily food intake. Fenofibrate dose (mg/kg/day) 0 Initial body weight (g) Final body weight (g) Liver weight (% of body weight) Food intake (g per group/day)
246 267 3.1 63
5 ± ± ± ±
8 13# 0.3 9
246 267 3.8 56
10 ± ± ± ±
7 9# 0.4 10
251 268 4.5 55
20 ± ± ± ±
11 23 0.2* 10
252 259 4.8 50
50 ± ± ± ±
2 11 0.4* 8*
249 229 5.3 37
± ± ± ±
12 17* 0.3* 12*
Data are presented as mean ± SEM (n = 4). Liver weight is expressed as a percent of body weight. Food intake is presented as daily food intake in g per tested group. * Significantly different from control group (p < 0.05). # Significantly different from initial body weight for respective group (p < 0.05).
slightly but significantly by 9% as comparing to initial body weight (p < 0.05). Final body weight of the groups receiving 10 and 20 mg/kg/day were 7% and 3% higher than at the beginning of the experiment (p > 0.05). On the contrary, the final body weight of animals treated with the highest dose decreased by 8% (p > 0.05). Final body weights of rats treated with 5 and 10 mg/kg/day fenofibrate dose were almost identical as control group. Animals given 20 and 50 mg/kg/day had lower final body weight by 3% and 14%, respectively. There was a significant difference between the group administered with 50 mg/kg/day of fenofibrate when compared to control as well as compared to rats treated with 5 and 10 mg/kg/day (p < 0.05). Liver weight (expressed as a percent of body weight) was dosedependently higher by 21%, 44%, 53% and 68% in fenofibrate-treated rats than in control group (p < 0.0001). The difference was statistically significant starting from the dose of 10 mg/kg/day. Daily food intake decreased in a dose-related manner and was significantly lower in rats given 20 and 50 mg/kg/day fenofibrate than in control group (p < 0.0001).
3.3. Fenofibrate effect on BDK protein expression Changes in BDK protein expression did not correspond with changes in BDK activity. There was not any significant difference in BDK protein expression between fenofibrate-treated groups and control rats (100%; 104%, 96% and 99% vs. 100% for control, respectively) (Fig. 2). 3.4. Fenofibrate effect on BCKDH activity In all fenofibrate-treated rats both actual and total BCKDH complex activities increased significantly in response to graded levels of administered drug starting from the lowest dose (Fig. 3). BCKDH actual activity was 3.7 ± 0.3, 4.1 ± 0.1, 4.6 ± 0.3 and 4.0 ± 0.3 fold higher than in control group (p < 0.0001). BCKDH total activity increased by 1.3 ± 0.1, 1.3 ± 0.1, 1.5 ± 0.1 and 1.3 ± 0.1 fold in animals receiving 5, 10, 20 or 50 mg/kg/day fenofibrate, respectively (p < 0.001). Consequently, BCKDH activity state (the percentage of active, dephosphorylated form) rats was 2.8 ± 0.2,
3.2. Fenofibrate effect on BDK activity Comparing to control group liver BDK activity decreased significantly in a dose-dependent manner by 61%, 64%, 66% and 89% in rats receiving 5, 10, 20 and 50 mg/kg/day of fenofibrate, respectively (p < 0.0001) (Fig. 1).
Fig. 1. Effect of increasing doses of fenofibrate on BDK activity in rat’s liver. Rats were fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Data are presented as mean ± SEM (n = 4). BDK activity is expressed as the first-order rate constant of ATP-dependent inactivation of BCKDH complex. *Significantly different from control group (p < 0.0001).
Fig. 2. Western blot analysis of BDK amounts in liver of rats fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Upper panel: representative immunoblot. Lower panel: bar graphs showing the expression level (arbitrary units-% of control) of BDK protein in each treatment group. Data are presented as mean ± SEM (n = 4).
4
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
Fig. 3. Effect of increasing doses of fenofibrate on BCKDH actual activity (A), total activity (B) and activity state (C) in rat’s liver. Rats were fed low-protein chow and administered increasing doses of fenofibrate or given only vehicle (control group—0 g/kg/day fenofibrate). Data are presented as mean ± SEM (n = 4). BCKDH total activity was determined after complete dephosphorylation of BCKDH complex with broad-specificity phosphatase. Activity state means percentage of BCKDH complex in active dephosphorylated state. *Significantly different from low-protein group (p < 0.0001). **Significantly different from low-protein group (p < 0.001).
3.1 ± 0.1, 3.2 ± 0.1 and 3.0 ± 0.1 fold higher than in control group (p < 0.0001). There were no statistical differences in actual and total BCKDH activity as well as BCKDH activity state among particular groups treated with different doses of fenofibrate (Fig. 3). 4. Discussion To our knowledge the dose-dependent effect of fenofibrate on BDK and BCKDH activities has not been studied yet. Results of the present study demonstrated that fenofibrate disturbed physiological regulation of BDK. Administration of increasing doses of fenofibrate to rats fed low-protein chow have a dose-related reduction in liver BDK activity. However changes in BDK protein expression did not correspond with changes in BDK activity. Therefore, it is possible that fenofibrate at tested doses affects BDK activity on posttranslational level. It was established that clofibrate and its active metabolite, clofibric acid regulate BDK activity by different mechanisms including direct inhibitory effect on BDK activity (Kobayashi et al., 2002). Clofibric acid is structurally related to ␣-ketoisocaproate, a physiological allosteric inhibitor of BDK (Paxton and Harris, 1984). Because fenofibrate similarly to clofibrate belongs to the group of fibric acid derivatives it is conceivable that in used doses it may inhibit BDK activity directly without changing the amount of BDK protein. In condition of reduced BDK activity BDP activity prevails and higher proportion of BCKDH E1␣ subunit occurs in an active dephosphorylated form what results in higher activity state of BCKDH. Significant increase in liver BCKDH activity state was observed with 5 mg/kg/day dose, and subsequent higher doses did not further increase significantly BCKDH activity state. Probably inhibition of BDK activity by the lowest tested dose of fenofibrate was sufficient to allow almost complete dephosphorylation of BCKDH E1␣ subunit by BDK. So further decrease in BDK activity caused by increasing doses of fenofibrate did not result in any significant increase in BCKDH activity state. Since one of the adverse effects of treatment with fenofibrate is myopathy (Hodel, 2002) it is worth considering the relationship between fenofibrate effect on BCKDH complex and myopathy. It is possible that that fenofibrate-induced up-regulation of BCKDH complex activity leads to reduction in BCAA levels which in turn may affect BCAA-regulated processes. BCAA that account for about
35% amino acids in muscle proteins play a special role in muscle metabolism (Shimomura et al., 2001). Leucine is thought to promote muscle protein anabolism not only by an increase in substrate availability. Leucine stimulates muscle protein synthesis by different mechanism including induction of both the activity and synthesis of proteins involved in mRNA translation (May and Buse, 1989; Crozier et al., 2005; Anthony et al., 2001). In addition, leucine inhibits muscle protein degradation (Louard et al., 1990; Nair et al., 1992). It was shown that BCAA catabolism enhanced by clofibrate treatment has significant influence on the leucineinduced activation of protein translation (Ishiguro et al., 2006). It cannot be excluded that fenofibrate disturbs regulation of muscle protein synthesis and/or degradation. Precisely balanced protein turnover is critical to maintain metabolic homeostasis. Even a small decrease in protein synthesis or acceleration of protein degradation can result in disturbances of physiological balance between these two crucial processes. Therefore, it is conceivable that the reduced levels of leucine may be one of the factors involved in pathomechanism of the myopathy observed during treatment with fenofibrate. Numerous studies that used animals fed different chows demonstrated a negative effect of fenofibrate on body weight, adiposity and food intake (Chaput et al., 2000; Mancini et al., 2001; Larsen et al., 2003). In the current study it was shown that fenofibrate effect on body weight was also present in rats fed low-protein chow. Fenofibrate starting from the dose of 10 mg/kg/day prevented body weight gain while daily food intake was reduced in rats administered with 20 and 50 mg/kg/day. This observation suggests that not only insufficient dietary food intake but also fenofibrateinduced metabolic changes could be the cause of the lack of body weight gain. Fenofibrate was shown to reduce food intake and increase resting energy expenditure by induction of the fatty acids catabolism and uncoupling proteins synthesis in rat liver (Mancini et al., 2001; Park et al., 2007). It is well documented that administration of fibrates to rodents results in numerous hepatic alterations, including hepatomegaly, an increase in the number and size of peroxisomes, and an increase in the expression of genes encoding peroxisomal, mitochondrial, and microsomal fatty acid-metabolizing enzymes (Balfour et al., 1990; Reddy and Chu, 1996). We observed a dose-related increase in the liver weight to body weight ratio in rats treated with fenofibrate starting from the dose of 10 mg/kg/day. Results of the present study demonstrated that fenofibrate at used doses induced hepatomegaly in dose-dependent manner in rats submitted to dietary protein restriction.
M. Knapik-Czajka et al. / Toxicology 266 (2009) 1–5
5. Conclusions It can be concluded that under condition of dietary protein restriction fenofibrate starting from the lowest dose inhibits BDK activity at posttranslational level and increases BCKDH complex activity state. That opposite to physiological regulation effect of fenofibrate may result in up-regulation of BCAA catabolism and decrease in their levels. Leucine, valine and isoleucine are essential amino acids which must be continuously available for body protein synthesis. In addition, leucine promotes muscle protein anabolism by different mechanisms. It cannot be excluded that the reduction of BCAA levels induced by up-regulation of their degradation is one of the factors involved in the pathomechanism of myopathy, one of most serious adverse effect observed during treatment with fenofibrate. Conflict of interest None declared. Acknowledgements This study was supported by State Committee for Scientific Research (KBN) grant number 3 P05F 032-25 (2003–2007). Authors would like to thank Jagoda Drag, MSc and Katarzyna Wadowska for their excellent technical support and Zuzanna Mikosza, MSc for her help in preparation of the manuscript. References Anthony, J.C., Anthony, T.G., Kimball, S.R., Jefferson, L.S., 2001. Signaling pathways involved in translational control of protein synthesis in skeletal muscle by leucine. J. Nutr. 131, 856–860. Balfour, J.A., McTavish, D., Heel, C., 1990. Fenofibrate. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in dyslipidaemia. Drugs 40, 260–290. Chapman, M.J., 2003. Fibrates in 2003: therapeutic action in atherogenic dyslipidaemia and future perspectives. Atherosclerosis 171, 1–13. Chaput, E., Saladin, R., Silvestre, M., Edgan, A.D., 2000. Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochem. Biophys. Res. Commun. 271, 445–450. Crozier, S.J., Kimball, S.R., Emmert, S.W., Anthony, J.C., Jefferson, L.S., 2005. Oral leucine administration stimulates protein synthesis in rat skeletal muscle. J. Nutr. 135, 376–382. Damuni, Z., Merryfield, M.L., Humphreysn, J.S., Reed, L., 1984. Purification and properties of branched-chain ( ketoacid dehydrogenase phosphatase from bovine kidney. Proc. Natl. Acad. Sci. U.S.A. 81, 4335–4338. Doering, C.B., Danner, D.J., 2000. Amino acid deprivation induces translation of branched-chain (-keto acid dehydrogenase kinase. Am. J. Physiol. Cell. Physiol. 279, 1587–1594. Espinal, R., Beggs, M., Patel, H., Randle, P.J., 1986. Effects of low-protein diet and starvation on the activity of branched-chain 2-oxo acid dehydrogenase kinase in rat liver and heart. Biochem. J. 237, 285–288. Goodwin, G.W., Zhang, B., Patton, R., Harris, R.A., 1988. Determination of activity and activity state of branched-chain (-ketoacid dehydrogenase in rat tissues. Methods Enzymol. 166, 189–201. Han, A.C., Goodwin, G.W., Paxton, R., Harris, R.A., 1987. Activation of branched chain (-ketoacid dehydrogenase in isolated hepatocytes by branched chain (ketoacids. Arch. Biochem. Biophys. 258, 85–94. Harris, R.A., Paxton, R., Parker, R.A., 1982. Activation of the branched-chain ␣ketoacid dehydrogenase complex by a broad specificity protein phosphatase. Biochem. Biophys. Res. Commun. 107, 1497–1503. Harris, R.A., Paxton, R., Powell, S.M., Goodwin, G.W., Kuntz, M.J., Han, A.C., 1986. Regulation of branched-chain ␣-ketoacid dehydrogenase complex by covalent modification. Adv. Enzyme Regul. 250, 219–237. Harris, R.A., Goodwin, G.W., Paxton, R., Dexter, P., Powell, S., Zhang, B., Han, A., Shimomura, Y., Gibson, R., 1996. Nutritional and hormonal regulation of the activity state of hepatic branched-chain ␣-ketoacid dehydrogenase complex. Ann. N. Y. Acad. Sci. 804, 307–313. Harris, R.A., Hawes, J., Popov, K.M., Zhao, Y., Shimomura, Y., Sato, J., Jaskiewicz, J., Hurley, T.D., 1997. Studies on the regulation of the mitochondrial ␣-ketoacid dehydrogenase complexes and their kinases. Adv. Enzyme Rev. 37, 271–293. Harris, R.A., Joshi, M., Jeoung, N.H., 2004. Mechanism responsible for regulation of branched-chain amino acid catabolism. Biochem. Bipohys. Res. Commun. 313, 391–396. Hodel, C., 2002. Myopathy and rhabdomyolysis with lipid-lowering drugs. Toxicol. Lett. 128, 159–168.
5
Honda, K., Ono, K., Mori, T., Kochi, H., 1991. Both induction and activation of the branched-chain 2-oxo acid dehydrogenase complex in primary-cultured rat hepatocytes by clofibrate. J. Biochem. 109, 822–827. Hutson, S.M., Lieth, E., LaNoue, K.F., 2001. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J. Nutr. 131, 846–850. Ijichi, C., Matsumura, T., Tsuji, T., Eto, Y., 2003. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem. Biophys. Res. Commun. 303, 59–64. Ishiguro, H., Katano, Y., Nakano, I., Ishigami, M., Hayashi, K., Honda, T., Goto, H., Bajotto, G., Maeda, K., Shimomura, Y., 2006. Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver. Life Sci. 79, 737–743. Knapik-Czajka, M., Cyganek, I., Tyszka-Czochara, M., Francik, R., Jaskiewicz, J., 2002. Effect of fibrates on the branched-chain ␣-ketoacid dehydrogenase complex in rat liver. Acta Pol. Toxicol. 10, 191–198. Knapik-Czajka, M., Tyszka-Czochara, M., Jaskiewicz, J., 2003. Effect of fibrates on the branched chain ␣-ketoacid dehydroganase complex activity. Acta Toxicol. 11, 59–65. Knapik-Czajka, M., Jaskiewicz, J., 2003. The influence of fibrates on the branchedchain ␣-ketoacid dehydrogenase complex expression in rat’s liver. Acta Toxicol. 11, 67–73. Kobayashi, R., Murakami, T., Obayashi, M., Nakai, N., Jaskiewicz, J., Fujiwara, Y., Shimomura, Y., Harris, R.A., 2002. Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms. Arch. Biochem. Biophys. 407, 31–240. Larsen, P.J., Jensen, P.B., Sorensen, R.V., Larsen, L.K., Vrang, N., Wulff, E.M., Wassermann, K., 2003. Differential influences of peroxisome proliferator-activated receptors ␥ and ␣ on food intake and energy homeostasis. Diabetes 52, 2249–2259. Li, C., Najafi, H., Daikhin, Y., Nissim, I.B., Collins, H.W., Yudkoff, M., Matschinsky, F.M., Stanley, C.A., 2003. Regulation of leucine-stimulated insulin secretion and glutamine metabolism in isolated rat islets. J. Biol. Chem. 278, 2853–2858. Louard, R.J., Barrett, E.J., Gelfand, R.A., 1990. Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man. Clin. Sci. 79, 457–466. Lu, G., Sun, H., She, P., Youn, J.Y., Warburton, S., Ping, P., Vondriska, T.M., Cai, H., Lynch, C.J., Wang, Y., 2009. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells. J. Clin. Invest. 119, 1678–1687. Mancini, F.P., Lanni, A., Sabatino, L., Moreno, M., Giannino, A., Contaldo, F., Colantuoni, V., Goglia, F., 2001. Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats. FEBS Lett. 491, 154–158. May, M.E., Buse, M.G., 1989. Effects of branched-chain amino acids on protein turnover. Diabetes Metab. Rev. 5, 227–245. Mitsubuchi, H., Owada, M., Endo, F., 2005. Markers associated with inborn errors of metabolism of branched-chain amino acids and their relevance to upper levels of intake in healthy people: an implication from clinical and molecular investigations on maple syrup urine disease. J. Nutr. 135, 1565–1570. Nair, K.S., Schwartz, R.G., Welle, S., 1992. Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. Am. J. Physiol. Endocrinol. Metab. 263, 928–934. Park, M.-K., Lee, H.-J., Hong, S.-H., Choi, S.-S., Yoo, Y.H., Lee, K.I., Kim, D.K., 2007. The increase in hepatic uncoupling by fenofibrate contributes to a decrease in adipose tissue in obese rats. J. Kor. Med. Sci. 22, 235–241. Paul, H.S., Liu, W.Q., Adibi, S.A., 1996. Alternation in gene expression of branchedchain ketoacid dehydrogenase kinase but not in gene expression of its substrate in the liver of clofibrated rat. Biochem. J. 317, 411–417. Paxton, R., Harris, R.A., 1984. Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain ␣-ketoacid dehydrogenase kinase in vitro and in perfused rat heart. Arch. Biochem. Biophys. 231, 58–66. Popov, K.M., Zhao, Y., Shimomura, Y., Jaskiewicz, J., Kedishvilli, N.Y., Irwin, J., Goodwin, G.W., Harris, R.A., 1995. Dietary control and tissue expression of branched-chain ␣-ketoacid dehydrogenase complex kinase. Arch. Biochem. Biophys. 316, 148–154. Reddy, J., Chu, R., 1996. Peroxisome proliferator-induced pleiotropic responses: pursuit of phenomenon. Ann N. Y. Acad. Sci. 804, 176–201. Schoonjans, K., Staels, B., Auwerx, J., 1996. Role of peroxisome proliferator-activated receptor [PPAR] in mediating the effects of fibrates and fatty acid on gene expression. J. Lipid Res. 37, 907–925. Shimomura, Y., Nanaumi, N., Suzuki, M., Popov, K.M., Harris, R.A., 1990. Purification and partial characterization of branched-chain ␣-ketoacid dehydrogenase kinase from rat liver and rat heart. Arch. Biochem. Biophys. 283, 293–299. Shimomura, Y., Obayashi, M., Murakami, T., Harris, R., 2001. Regulation of branchedchain amino acid catabolism: nutritional and hormonal regulation of activity and expression of the branched-chain ␣-keto acid dehydrogenase kinase. Curr. Opin. Clin. Nutr. Metab. Care 4, 419–423. Wynn, R.M., Davie, J.R., Chuang, D.T., 1996. Structure, function and assembly of mammalian branched-chain ␣-keto acid dehydrogenase complex. In: Patel, M.S., Roche, T.E., Harris, R.A. (Eds.), Alpha-keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel, Switzerland, pp. 101–118. Zhao, Y., Hawes, J., Popov, K.M., Jaskiewicz, J., Shimomura, Y., Crabb, D.W., Harris, R.A., 1994a. Site directed-mutagenesis of phosphorylation sites of the branchedchain ␣-ketoacid dehydrogenase complex. J. Biol. Chem. 269, 18583–18587. Zhao, Y., Hawes, J., Popov, K.M., Jaskiewicz, J., Shimomura, Y., Kedishvilli, N.Y., Jaskiewicz, J., Kuntz, M.J., Kain, J., Hang, B., Harris, R.A., 1994b. Effect of dietary protein on the liver content and subunit composition of the branched-chain ␣-ketoacid dehydrogenase complex. Arch. Biochem. Biophys. 308, 446–453.