Acute effects of phenylbutyrate on glutamine, branched‐chain amino acid and protein metabolism in skeletal muscles of rats

Summary

Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed in vivo and in vitro experiments to examine the effects of PB on glutamine (GLN), branched‐chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5 mM). L‐[1‐¹⁴C] leucine was used to estimate protein synthesis and leucine oxidation, and 3‐methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched‐chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.

4‐Phenylbutyric acid (PB) is an aromatic fatty acid that acts as a chemical chaperone and histone deacetylase inhibitor. The compound is under investigation for use in treatment of cystic fibrosis, glioma, acute myeloid leukaemia, sickle cell disorders, amyotrophic lateral sclerosis, Wilson's disease, Huntington's disease and other disorders (Iannitti & Palmieri 2011). PB is not known to be mutagenic, carcinogenic or teratogenic. The reports of some adverse events, which most commonly included nausea, emesis, weakness, dizziness, gait instability, memory loss and headache, were rapidly reversible after reduction or interruption of PB dosing (Perrine et al. 2011; Mokhtarani et al. 2013). Therefore, PB is accepted as safe and well‐tolerated agent with potential of clinical use in treatment of lethal illnesses that do not have any effective treatment (Iannitti & Palmieri 2011).

However, some adverse effects of PB have been observed, and these are supposed to be due to its effect on amino acid metabolism, particularly on glutamine (GLN) and branched‐chain amino acids (BCAA; valine, leucine and isoleucine). PB is converted in vivo by β‐oxidation into phenylacetate that is rapidly conjugated with GLN to form phenylacetylglutamine, which is excreted in the urine. Some reports of the use of PB in treatment of hyperammonaemia in urea cycle disorders demonstrate that PB decreases concentration of GLN and BCAA in blood plasma (Brunetti‐Pierri et al. 2011), PB decreased GLN and BCAA concentrations in the blood plasma in hepatectomized and laparotomized rats (Holecek & Vodenicarovova 2016). Both GLN and BCAA act as anabolic agents and play important role in many processes including cell proliferation, expression of genes, and hormone production. Thus one could suppose that PB treatment could result in severe deficiency of GLN and BCAA, which may worsen the course of the illness, particularly via suppression of immune functions, impaired gut integrity and altered protein metabolism in skeletal muscle.

The first aim of this study was to assess the effect of PB treatment on GLN and BCAA concentrations in blood plasma and muscles. The second aim was to examine the effects of PB on GLN, BCAA and protein metabolism in two types of skeletal muscle of different metabolic properties – soleus (SOL, slow‐twitch, red muscle) and extensor digitorum longus (EDL, fast‐twitch, white muscle). The studies were performed both under in vivo and in vitro conditions to examine if the observed effects of PB are mediated directly or indirectly.

Materials and methods

Animals and material

Male Wistar rats (BioTest, Konarovice, CR) were housed in standardized cages in quarters with controlled temperature and a 12‐h light–dark cycle. Rats were maintained on a ST‐1 (Velas, CR) standard laboratory diet containing (w/w) 24% of nitrogenous compounds, 4% fat, 70% carbohydrates and 2% of minerals and vitamins, and drinking water ad libitum.

Sodium 4‐phenylbutyrate (PB) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Chemicals were obtained from Sigma Chemical (St. Louis, MO, USA), Lachema (Brno, CR), Waters (Milford, MA, USA), Biomol (Hamburg, Germany) and Merck (Darmstadt, Germany).

Ethical approval statement

The Animal Care and Use Committee of Charles University, Faculty of Medicine in Hradec Kralove specifically approved this study (Licence No. 144879/2011‐MZE‐17214). All applicable international, national, local and/or institutional guidelines governing the use of experimental animals were followed. Animals were treated carefully by animal experts educated and trained on how to manipulate animals to maintain a healthy environment and to reduce distress and minimize potential pain and suffering.

Experimental design and methods

Two series of experiments have been performed: the first under in vivo conditions and the second under in vitro conditions.

Study 1. Effects of PB under in vivo conditions

Rats weighing approximately 250 g each received three consecutive intraperitoneal injections of PB (300 mg/kg b.w.) or saline at 12‐h intervals (Davies et al. 2009). To exclude nutritional effects, rats fasted during the study. The animals were sacrificed 1 h after the last injection in pentobarbital narcosis by exsanguination from the abdominal aorta. Blood was collected in heparinized tubes and immediately centrifuged for 10 min at 2000 × g using a refrigerated centrifuge, and blood plasma was transferred into clean polypropylene tubes using a Pasteur pipette. Afterwards, small pieces (approximately 0.1 g) of soleus (SOL) and extensor digitorum longus (EDL) muscles were quickly removed and frozen in liquid nitrogen.

Amino acid concentrations

Amino acid concentrations were determined in supernatants of deproteinized samples of blood plasma, SOL, and EDL by liquid chromatography (Alliance 2695, Waters, Milford, MA, USA) after derivatization with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (Waters, Milford, MA, USA) using norleucine as internal standard. The intracellular concentration of each amino acid was calculated by subtracting the free extracellular part from the total amount assuming the plasma concentration to be equal to the concentration in the interstitial fluid as described by Bergström et al. (1974). Total tissue water was measured from the tissue weight obtained after drying for 24 h at 90°C. The determination of extra‐ and intracellular water was based on the chloride method according to Graham et al. (1967).

Branched‐chain keto acid (BCKA) concentrations

The reversed‐phase HPLC combined with o‐phenylenediamine derivatization was used for determination of BCKA in samples of blood plasma and incubation medium. Samples were pH‐adjusted with 6 M sodium acetate (pH 6.0) prior to the chromatographic analysis. Keto acids were separated on LichroCart 125 × 4 mm, Purospher Star RP‐18 (5 μm) endcapped analytical column (Merck Millipore, Darmstadt Germany) using mobile phase consisting of methanol and water at flow rate 0.8 ml/min in a gradient mode. The quinoxalinol derivatives of BCKAs were detected using fluorescence with emission and excitation at 410 nm and 350 nm, respectively, and quantified by internal standard method.

Glutamine synthetase activity

Glutamine synthetase activities in muscles were determined using a colorimetric assay based on the formation of γ‐glutamyl‐hydroxamate (Minet et al. 1997). Tissue samples were homogenized in imidazole buffer (pH 6.8, 0°C) with excess of GLN and hydroxylamine (1/9 w/v) and incubated at 37°C for 30 min. After centrifugation, the colour created by reaction with ferric chloride was measured at 530 nm in Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Switzerland). The results were expressed as relative units obtained by normalizing of the glutamine synthetase activity for control of SOL (A_SOL = 1).

Study 2. Effects of PB on muscle under in vitro conditions

Animals weighing approximately 50 g were sacrificed in pentobarbital narcosis (6 mg/100 g body weight, intraperitoneally) by exsanguination via abdominal aorta. SOL and EDL muscles of both legs were dissected according to Maizels et al. (1977). Young animals weighing less than 75 g must be used to obtain muscles for in vitro experiments to avoid metabolic changes attributable to inadequate diffusion of gases and metabolites between the tissue and the medium (Maizels et al. 1977). Muscles were fixed via the tendons to stainless steel clips to provide slight tension (at approximately resting length) and immediately transferred into 2.5 ml of modified Krebs–Henseleit bicarbonate buffer (in mM: 144 Na⁺, 4.2 K⁺, 0.8 Mg²⁺, 1.3 Ca²⁺, 123 Cl⁻, 26 HCO³⁻, 0.8 SO₄ ²⁻, 2 H₂PO4⁻) with 6 mM glucose, 2.77 mM amino acids and 2 mU/ml insulin (pH 7.4, 37 ^°C) saturated with O₂/CO₂ (19:1). The viability of the incubated muscles was previously confirmed in our laboratory by comparing the differences of energy charge ([ATP + ADP/2]/[ATP + ADP + AMP]), wet/dry weight ratio, and values of protein synthesis, leucine oxidation and proteolysis before and after incubation (Safranek et al. 2003a,b). SOL and EDL of the right leg were used for determination of the effect of PB, and muscles of the left leg served as paired controls.

The muscles were pre‐incubated for 30 min in a thermostatically controlled bath (37°C) with a shaking device (70 cycles/min) to ensure stable intramuscular concentrations of components present in the medium. After the pre‐incubation, the muscles were quickly rinsed in 0.9% saline solution, blotted and transferred to a second set of vials and incubated in a medium containing 5 mM PB or in the same medium without PB. Concentration of PB in medium was based on the study of Carducci et al. (1996) who observed induction of apoptosis in human prostate cancer cells. Other components present in the medium were dependent on the parameter measured as described below.

Leucine oxidation and protein synthesis in muscles (in vitro study)

Leucine oxidation and protein synthesis were determined using L‐[1−¹⁴C]leucine as previously described in detail (Safranek et al. 2003a,b). Leucine oxidation rates were calculated using the radioactivity of released ¹⁴CO₂, efficiency of ¹⁴CO₂ recovery and leucine‐specific activity in the incubation medium. The results were expressed as nmol of oxidized leucine/mg muscle protein per hour. Protein synthesis rates were calculated using the radioactivity of leucine incorporated in muscle protein and leucine‐specific activity in the incubation medium. The results were expressed as nmol of incorporated leucine/mg protein per hour. The protein content was measured according to Lowry et al. (1951).

3‐Methylhistidine (3‐MH) release into incubation medium

Concentration of 3‐MH, a characteristic product of the myofibrillar breakdown, in incubation medium was quantified using a high‐performance liquid chromatography method (Alliance 2695, Waters, Milford, MA, USA) based on the reaction with fluorescamine. The release of 3‐MH from incubated muscles was calculated on the basis of its concentrations in medium after two hours of the incubation, the volume of the medium and the weight of the muscle. Results were expressed as nmol/g wet muscle per hour.

Statistical analysis

The results are expressed as mean±SE. F‐test followed by unpaired t‐test (to estimate the effect of PB under in vivo conditions) and by paired t‐test (to estimate the effect of PB on paired muscles from the same animal under in vitro condition) has been used for analysis of the data. Differences were considered significant at P ˂ 0.05. Statistical software NCSS 2001 (Kaysville, UT, USA) was applied for analysis.

Results

Effects of PB on GLN and BCAA concentrations (study in vivo)

PB treatment decreased GLN and BCAA, that is valine, isoleucine and leucine concentrations in blood plasma. The decrease in the BCAA in blood plasma was more significant than the decrease in GLN and was found also in muscles while GLN concentration in muscles increased (Figure 1). The alterations in GLN and BCAA levels resulted in a marked increase in GLN to BCAA ratios both in blood plasma and muscles (Figure 2).

Figure 1.

Effects of PB on GLN and BCAA concentrations in blood plasma and in SOL and EDL muscles. Means ± SE of 10 animals, *P ˂ 0.05. Amino acid concentrations are given in μmol/l of blood plasma or mmol/l of intracellular fluid.

Figure 2.

Effects of PB on GLN to BCAA ratio in blood plasma and muscles. Means ± SE of 10 animals, *P ˂ 0.05.

Effects of PB on GLN and BCAA concentrations in incubation medium and in incubated muscles (study in vitro)

Addition of PB into incubation medium increased GLN and decreased BCAA concentrations in medium with EDL and increased GLN to BCAA ratio in medium of both muscles. The effect of PB of GLN concentration in incubation medium with SOL was insignificant (Table 1).

Table 1.

GLN and BCAA concentrations in incubation medium at the end of incubation

	SOL (μmol/l)		EDL (μmol/l)
	Saline (n = 12)	PB (n = 12)	Saline (n = 12)	PB (n = 12)
Gln (0)	87 ± 3	95 ± 5	60 ± 2	76 ± 8a
Val (113)	116 ± 1	117 ± 1	112 ± 1	110 ± 1a
Ile (82)	82 ± 1	82 ± 1	80 ± 1	77 ± 1a
Leu (162)	163 ± 2	163 ± 2	158 ± 2	154 ± 2a
BCAA (358)	361 ± 4	363 ± 3	350 ± 5	341 ± 4a
GLn/BCAA	0.24 ± 0.01	0.26 ± 0.01a	0.17 ± 0.01	0.22 ± 0.02a

Means ± SE.

^a P ˂ 0.05 (effect of PB in specific muscle type, paired t‐test).

Amino acid concentrations at the beginning of incubation are reported in parentheses.

Addition of PB into medium increased GLN concentrations in both muscles and GLN/BCAA in EDL. The effect of PB on intramuscular concentrations of the BCAA was insignificant (Table 2).

Table 2.

GLN and BCAA concentrations in muscles at the end of incubation

	SOL (mmol/g)		EDL (mmol/g)
	Saline (n = 12)	PB (n = 12)	Saline (n = 12)	PB (n = 12)
Gln	4.80 ± 0.26	5.48 ± 0.26a	4.65 ± 0.14	5.37 ± 0.26a
Val	0.29 ± 0.02	0.31 ± 0.02	0.20 ± 0.01	0.21 ± 0.01
Ile	0.21 ± 0.01	0.20 ± 0.01	0.14 ± 0.01	0.13 ± 0.01
Leu	0.35 ± 0.03	0.36 ± 0.02	0.21 ± 0.01	0.22 ± 0.01
BCAA	0.85 ± 0.06	0.87 ± 0.05	0.55 ± 0.02	0.56 ± 0.03
Gln/BCAA	5.85 ± 0.49	6.56 ± 0.57	8.62 ± 0.41	9.80 ± 0.68a

Means ± SE.

^a P ˂ 0.05 (effect of PB in specific muscle type, paired t‐test).

Effects of PB on leucine oxidation and protein metabolism in skeletal muscle (study in vitro)

Addition of PB to incubation medium increased leucine oxidation in both muscle types, decreased protein synthesis in SOL and enhanced protein breakdown in EDL (Figure 3). The rise in leucine oxidation was more pronounced in EDL (55%) than in SOL (29%).

Figure 3.

Effects of PB on leucine oxidation, protein synthesis and proteolysis in incubated muscles. Means ± SE of values obtained from 12 animals, *P ˂ 0.05.

Effects of PB on BCKA concentrations

A marked decrease in all three BCKAs was found in blood plasma of PB‐treated animals (Figure 4) and in incubation medium containing PB (Table 3).

Figure 4.

Effects of PB on BCKA concentrations in blood plasma. Mean ± SE of 10 animals, *P ˂ 0.05.

Table 3.

BCKA concentrations in incubation medium at the end of incubation

	SOL (μmol/l)		EDL (μmol/l)
	Saline (n = 12)	PB (n = 12)	Saline (n = 12)	PB (n = 12)
KIV	1.00 ± 0.05	0.18 ± 0.00a	1.19 ± 3	0.20 ± 0.01a
KIC	2.25 ± 0.11	0.79 ± 0.02a	2.58 ± 0.17	0.79 ± 0.03a
KMV	1.23 ± 0.06	0.62 ± 0.04a	1.94 ± 0.07	1.20 ± 0.03a
BCKA	4.48 ± 0.21	1.59 ± 0.05a	5.71 ± 0.31	2.19 ± 0.05a

Means ± SE.

^a P ˂ 0.05 (effect of PB in specific muscle type, paired t‐test).

KIV: α‐ketoisovalerate (ketovaline); KIC: α‐ketoisocaproate (ketoleucine); KMV: α‐keto‐β‐methylvalerate (ketoisoleucine).

Effects of PB on GLN synthesis in muscles

Figure 5 shows that glutamine synthetase activity was higher in EDL compared with SOL and that PB treatment increased glutamine synthetase activity in both muscle types.

Figure 5.

Effects of PB on glutamine synthetase activities in skeletal muscle. Means ± SE of 10 animals, *P ˂ 0.05. Relative units were obtained by normalizing of the glutamine synthetase activity for SOL of saline treated control (A_SOL = 1).

Discussion

Studies demonstrate that GLN deficiency impairs immune functions, gut integrity and protein balance in skeletal muscle (Buchman 1999; Exner et al. 2002; Hardy & Hardy 2008; Holecek & Sispera 2014). The BCAA are essential amino acids, which serve as an essential substrate and regulator in protein synthesis (Tischler et al. 1982; Kimball & Jefferson 2001; Nair & Short 2005). Therefore, the decrease in GLN and BCAA levels after PB administration may lead to various adverse effects on the body, which may have disastrous consequences particularly in patients with severe illness, in which GLN concentrations in blood plasma are frequently low and BCAA catabolism is activated (Holecek et al. 1997; Exner et al. 2002; Hardy & Hardy 2008).

While the decrease in GLN in blood plasma of PB‐treated animals is undoubtedly due to the formation of phenylacetyl‐GLN and its elimination by the urine, the mechanism causing the decrease in the BCAA is not completely clear. One possibility is that increased GLN disposal induces adaptive response characterized by enhanced consumption of BCAA for synthesis of glutamate that reacts with ammonia to GLN (Figure 6). This possibility has been confirmed in recent studies demonstrating that extracellular GLN deficiency enhances GLN release from muscle and activates BCAA catabolism while BCAA oxidation in muscles incubated in medium with high GLN concentration is inhibited (Holecek & Sispera 2014; Holecek et al. 2015). However, increased GLN concentrations and leucine oxidation in muscles incubated with PB suggest that the cause of BCAA deficiency in blood and muscles after PB treatment is different.

Figure 6.

Scheme demonstrating the role of BCAA in GLN synthesis and the supposed effects of PB administration on GLN and BCAA metabolism. 1, BCAA aminotransferase; 2, BCKA dehydrogenase; 3, glutamine synthetase.

An increase in leucine oxidation in muscles incubated in medium with PB indicates that PB activates the BCKA dehydrogenase, the rate‐limiting enzyme of the BCAA oxidation. The consecutive increase in the BCAA oxidation should lead to the observed decrease in the BCAA in muscles and in blood. The explanation is supported by report of Brunetti‐Pierri et al. (2011), who demonstrated using liver extracts from PB‐treated mice that PB enhances the proportion between active (unphosphorylated) and inactive (phosphorylated) form of the BCKA dehydrogenase by preventing phosphorylation of E1α subunit of the enzyme. It has been suggested that the stimulatory effect of PB on BCKA dehydrogenase activity may be used in treatment of maple syrup urine disease, a metabolic disorder caused by a deficiency of the BCKA dehydrogenase complex (Brunetti‐Pierri et al. 2011).

A consequence of activated oxidation of BCAA in muscles is the decrease in BCKA, which was observed both under in vivo (in blood plasma) and in vitro (in incubation medium) conditions. The BCKAs exert regulatory effects on protein metabolism in muscle, gluconeogenesis and ureagenesis in hepatocytes, insulin and glucagon release, act as an important source of energy and/or are reaminated to the BCAA (Tischler et al. 1982; Walser 1984; Holecek et al. 1998; Holecek 2001). However, the effects of decreased BCKA levels on the body are obscure.

Enhanced catabolism of the BCAA in muscle is connected with enhanced production of glutamate (Figure 6), which may be amidated by glutamine synthetase to GLN. Glutamine synthetase is regulated by a number of variables, including its substrates and products (Stadtman 2001), and it may be supposed that enhanced glutamate production and lack of feedback inhibition by end products of GLN metabolism due to enhanced GLN withdrawal as phenacetylglutamine have a role in activation of glutamine synthetase in PB‐treated animals. Nevertheless, a direct effect of PB on glutamine synthetase activity cannot be excluded.

Increased oxidation of the BCAA and GLN synthesis in muscles may have adverse effects both on protein synthesis and proteolysis. The inhibitory effect of PB on protein synthesis was more pronounced in SOL while proteolysis increased significantly in EDL. In this connection, it should be noted that the effects of PB on leucine oxidation and GLN concentration in muscles were more pronounced in EDL and the decrease in BCAA concentrations in incubation medium after addition of PB was observed only in medium with EDL. Some articles indicate associations between BCAA and protein metabolism in muscle (O'Donnel et al. 1976; Holecek 2011) and, therefore, the differences in BCAA and GLN metabolism induced by PB in SOL and EDL may play a role in different alterations in protein metabolism. The findings of more pronounced effects of PB in EDL (composed predominantly of white, fast‐twitch fibres) compared with SOL (composed predominantly of red, slow‐twitch fibres) is a further evidence of higher sensitivity of fast‐twitch fibres to various signals than slow‐twitch fibres as reported in number of other articles (Kadlcikova et al. 2004; Muthny et al. 2008, 2009).

In conclusion, to the best of our knowledge, this is the first study reporting that PB treatment decreases BCAA and BCKA levels and increases BCAA oxidation and GLN synthesis in skeletal muscle. These effects may exert adverse effects on protein balance in skeletal muscle and the course of the illness in which PB is used as a therapeutic agent. The results also indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be perspective in management of high concentrations of BCKA in maple syrup urine disease, a hereditary disease that is due to the lack of the BCKA dehydrogenase. Further studies are needed to examine the effects of chronical administration of PB on amino acid concentrations and protein balance.

Author contributions

The authors meet ICMJE authorship criteria, and nobody who qualifies for authorship has been excluded. M.H. provided conception and design of the study, interpreted results of experiments and prepared the manuscript. M.V. performed the experiments, analysed data and edited the manuscript, P.S. analysed data and edited manuscript.

Conflict of interest

The authors declared no potential conflict of interests with respect to the research, authorship and/or publication of this article.

Funding source

This work was supported by grants PRVOUK P37/02 and PROGRES Q40/02 of the Charles University.