Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver
Introduction
The translation phase in protein synthesis comprises four sequential steps, namely initiation, elongation, termination, and reuse of ribosome. Initiation is the most important step in the regulation of translation (Pain, 1996). It has been reported that, after starvation, food intake stimulates protein synthesis in the liver and skeletal muscle of rats (Yoshizawa et al., 1998) and that food intake is critical for stimulation of translation initiation (Yoshizawa et al., 1997). The amino acids from dietary protein, especially leucine, are known activators of translation initiation factors (Anthony et al., 2000, Hong and Layman, 1984, Kimball et al., 1998). It has also been reported that branched-chain amino acids (BCAAs) promote albumin synthesis in rat primary hepatocytes through the mammalian target of rapamycin (mTOR) signaling pathway with no effect on the synthesis of intracellular proteins and the amount of albumin mRNA (Ijichi et al., 2003).
Regulation of translation initiation occurs principally by changes in the phosphorylation state of eukaryotic initiation factors (eIFs) (Proud, 2002). Phosphorylation of eIF4E-binding protein-1 (4E-BP1) releases eIF4E, which is then free to bind with eIF4G to form eIF4F, the complex required for translation initiation. This occurs because the binding site for 4E-BP1 on eIF4E overlaps with the eIF4G binding site. Thus, 4E-BP1 plays a key role in the regulation of mRNA translation in eukaryotic cytoplasm (Flynn and Proud, 1996, Gingras et al., 1999). In addition, leucine has been reported to stimulate phosphorylation of 4E-BP1 in various organs (Fox et al., 1998, Kimball et al., 1998, Xu et al., 1998).
Phosphorylation of the ribosomal protein S6 catalyzed by specific kinases, especially S6 kinase 1 (S6K1), results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5′-end of the message (TOPS mRNAs), which include some elongation factors such as eEF1A and eEF2 (Kimball et al., 1999). Therefore, the S6K1 is suggested to play a key role in the regulation of protein synthesis by controlling the biosynthesis of translational components (Dufner and Thomas, 1999). The mTOR catalyzes phosphorylation of three sites on S6K1, namely Thr229, Thr389, and Ser404. Among these sites, phosphorylation of Thr389 plays the most important role in activation of S6K1 (Pearson et al., 1995).
Recent studies have demonstrated that mTOR mediates the activating effects of amino acids on 4E-BP1 and S6K1 (Kimball et al., 1999, Patti et al., 1998) and that mTOR Ser2448 phosphorylation is involved in the regulatory mechanism (Bolster et al., 2002, Peterson et al., 2000). Leucine is known to bring about activation of mTOR, which in turn signals phosphorylation of 4E-BP1 and S6K1. Time course studies on the changes in the phosphorylation state of 4E-BP1 and S6K1 induced by administration of leucine demonstrated that phosphorylation of these compounds in rat liver and muscle was at maximum levels 1 h after leucine loading (Yoshizawa et al., 2001).
Mammals have a characteristic catabolic system for leucine. The first step in this catabolic pathway is reversible transamination to form α-ketoisocaproate, which is catalyzed by the enzyme branched-chain aminotransferase (BCAT). Transamination of leucine occurs out of the liver, mainly in muscle, because BCAT is not found in adult rat liver (Hutson, 1989). The second step in the catabolism of leucine is irreversible oxidative decarboxylation of α-ketoisocaproate, which is catalyzed by the branched-chain α-keto acid dehydrogenase complex (BCKDC). This reaction is the rate-limiting step in the catabolism of leucine (Harper et al., 1984, Harris et al., 1994) and occurs mainly in the liver because the BCKDC activity is markedly higher in liver than in other organs (Harris et al., 1990). This organ specificity of enzymes that regulate BCAA catabolism is typical in rats (Suryawan et al., 1998).
The BCKDC activity is tightly controlled by a specific kinase (Shimomura et al., 1990a) and a specific phosphatase (Damuni and Reed, 1987). The kinase phosphorylates the E1 subunit of the complex, turning it inactive, and the phosphatase dephosphorylates E1 and reactivates the complex. Especially, the kinase plays a central role in the regulation of the BCKDC activity (Shimomura et al., 2001).
Clofibrate is a therapeutic agent for patients with hyperlipidemia. It has been reported that clofibric acid, an active metabolite of clofibrate, activates BCKDC and therefore increases the rate of leucine oxidative disposal (Kobayashi et al., 2002, Ono et al., 1990, Paul and Adibi, 1979, Paxton and Harris, 1984a). In addition, clofibric acid has been reported to inhibit protein synthesis (Paul and Adibi, 1980). These facts suggest that enhanced BCAA catabolism by clofibrate treatment might blunt signaling for protein synthesis. In the present study, we have examined this hypothesis using rats, and the liver was chosen as a target organ, since it contains a very high content of BCKDC.
Section snippets
Materials
Rabbit polyclonal antibodies against phospho-mTOR (Ser2448), total-mTOR, and phospho-S6K1 (Thr389) were purchased from Cell Signaling Technology (Beverly, MA, USA), and goat anti-4E-BP1 and rabbit anti-S6K1 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Enhanced chemiluminescence (ECL) reagents and Immobilon-P polyvinylidene difluoride (PVDF) membrane were purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, U.K.) and Millipore (Bedford, MA, USA),
Concentrations of plasma leucine, isoleucine, valine, and total BCAAs
Leucine administration to control rats increased plasma leucine concentration by ∼ 4-fold and markedly decreased plasma isoleucine and valine concentrations (Table 1). Clofibrate-saline treated rats had significantly decreased plasma BCAA concentrations; individual and total BCAA concentrations in the clofibrate–saline group were only less than 50% of those in the corresponding control group (Table 1). Leucine administration to clofibrate-treated rats significantly increased plasma leucine
Discussion
In the present study, leucine administration to control rats significantly increased plasma leucine concentrations and the phosphorylation state of hepatic mTOR, 4E-BP1, and S6K1, indicating that leucine promotes protein synthesis in the liver, as reported previously (Anthony et al., 2001, Ijichi et al., 2003). Leucine administration also significantly activated hepatic BCKDC, presumably by inhibiting the specific kinase for BCKDC (BDK) through α-ketoisocaproate, a natural BDK inhibitor derived
Acknowledgements
This work was in part supported by a Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (17300208 to YS).
References (48)
- et al.
Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation
Journal of Nutrition
(2000) - et al.
Oral administration of leucine stimulates ribosomal protein mRNA translation but not global rates of protein synthesis in the liver of rats
Journal of Nutrition
(2001) - et al.
AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling
Journal of Biological Chemistry
(2002) - et al.
Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria
Journal of Biological Chemistry
(1987) - et al.
Ribosomal S6 kinase signaling and the control of translation
Experimental Cell Research
(1999) - et al.
Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids
Archives of Biochemistry and Biophysics
(1987) - et al.
Regulation of the branched-chain alpha-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease
Advances in Enzyme Regulation
(1990) - et al.
Regulation of branched-chain amino acid catabolism
Journal of Nutrition
(1994) - et al.
Effects of liver failure on branched-chain alpha-keto acid dehydrogenase complex in rat liver and muscle: comparison between acute and chronic liver failure
Journal of Hepatology
(2004) - et al.
Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system
Biochemical and Biophysical Research Communications
(2003)
Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209
Journal of Biological Chemistry
Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myoblasts
Journal of Biological Chemistry
Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6
Journal of Biological Chemistry
Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms
Archives of Biochemistry and Biophysics
Nutritional supplementation with branched-chain amino acids in advanced cirrhosis: a double-blind, randomized trial
Gastroenterology
Determination of branched-chain alpha-keto acid dehydrogenase activity state and branched-chain alpha-keto acid dehydrogenase kinase activity and protein in mammalian tissues
Methods in Enzymology
Regulation by induction of branched-chain 2-oxo acid dehydrogenase complex in clofibrate-fed rat liver
Biochemical and Biophysical Research Communications
Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain alpha-ketoacid dehydrogenase kinase in vitro and in perfused rat heart
Archives of Biochemistry and Biophysics
Regulation of branched-chain alpha-ketoacid dehydrogenase kinase
Archives of Biochemistry and Biophysics
FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions
Journal of Biochemistry
The modular phosphorylation and activation of p70s6k
FEBS Letters
AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet
Journal of Nutrition
Clinical comparison of branched-chain amino acid (l-leucine, l-isoleucine, l-valine) granules and oral nutrition for hepatic insufficiency in patients with decompensated liver cirrhosis (LIV-EN study)
Hepatology Research
Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart
Archives of Biochemistry and Biophysics
Cited by (22)
Octanoic acid promotes branched-chain amino acid catabolisms via the inhibition of hepatic branched-chain alpha-keto acid dehydrogenase kinase in rats
2015, Metabolism: Clinical and ExperimentalCitation Excerpt :Known mechanisms for acute control of the activity of BCKDC include direct inhibition of the activity of the complex by NADH and CoA esters derived from the BCAAs [3], and activation of the complex can also be achieved in the short-term by inhibition of BDK activity by α-ketoisocaproate, the transamination product of leucine [4]. Moreover, α-chloroisocaproate [5] and clofibric acid [6–9] also promote activation of BCKDC by inhibition of BDK. Since total free fatty acid (FFA) and BCAA concentration in blood is increased in type 2 diabetes mellitus [10,11], there are many reports determined the relationship between FFA or FFA oxidation and BCAA catabolism [11,12].
Simvastatin increases liver branched-chain α-ketoacid dehydrogenase activity in rats fed with low protein diet
2014, ToxicologyCitation Excerpt :It has been demonstrated that specific conditions characterized by an increased liver BCKDH activity state such as clofibrate treatment or liver cirrhosis are associated with a reduced plasma BCAAs level (Honda et al., 2004; Kadota et al., 2012). Moreover Ishiguro et al. (2006) found that stimulation of liver BCKDH and BCAAs degradation by clofibrate had a negative effect on leucine-regulated muscle protein synthesis in rats. Additionally, muscle energy metabolism is altered in patients with liver cirrhosis and BCAAs administration alleviates this abnormality (Doi et al., 2004).
Stimulation of rat liver branched-chain alpha-keto acid dehydrogenase activity by low doses of bezafibrate
2013, ToxicologyCitation Excerpt :Kadota et al. (2012) proved that activation of the liver BCKDH complex by clofibrate resulted in a substantial decrease of plasma BCAA concentration. It was also found that clofibrate by stimulating liver BCKDH and BCAA catabolism impairs leucine-regulated muscle protein synthesis in rats (Ishiguro et al., 2006). It is conceivable that bezafibrate-induced stimulation of liver BCKDH activity leads to reduction in plasma BCAA levels which in turn may affect BCAA-regulated processes in muscle.
Biological functions and research progress of eIF4E
2023, Frontiers in Oncology