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Articles

Protein Expressions of Branched-Chain Keto Acid Dehydrogenase Subunits Are Selectively and Posttranscriptionally Altered in Liver and Skeletal Muscle of Starved Rats¹

Clinical Nutrition Research Unit, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

²To whom correspondence should be addressed at UPMC Health System, 200 Lothrop Street, MUH E-321, Pittsburgh, PA 15213. E-mail: adibi{at}msx.dept-med.pitt.edu

	ABSTRACT

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Although it has been well established that starvation increases the oxidation of branched-chain keto acids (BCKA) in humans and experimental animals such as rats, the mechanism has not been adequately investigated. For example, the effects of starvation on protein and mRNA expressions of BCKA dehydrogenase, which is the key enzyme regulating this oxidation, have not yet been studied. To initiate such studies, we first determined the activity of BCKA dehydrogenase in the liver and skeletal muscle of fed and starved rats. The levels of activity of BCKA dehydrogenase were significantly greater in tissues of starved rats than in those of fed rats. We then investigated the possible mechanisms of these increases in enzyme activity. The activity state of the enzyme was greater by 3-fold in the muscle of starved compared with fed rats, but there was no significant difference between the activity states in the liver. There were no significant differences between protein expressions of BCKA dehydrogenase subunits (E₁, E₁ß and E₂) in tissues of fed and starved rats; the exceptions were a greater expression of E₁ in the liver and a lower expression of E₁ß in the skeletal muscle of starved rats. These differences in protein expressions were not accompanied with any difference in the mRNA expressions of genes encoding E₁ and E₁ß. The rate of inactivation of BCKA dehydrogenase, mediated by its associated kinase, was significantly slower in the skeletal muscle of starved rats but was the same in the liver. However, there was no significant difference between the protein or the mRNA expressions of the gene encoding BCKA dehydrogenase kinase in tissues of fed and starved rats. These results show that starvation increases the activity of BCKA dehydrogenase in the liver and skeletal muscle, and the mechanisms of increases in activity are posttranscriptional and involve cellular rather than the molecular mechanisms.

KEY WORDS: • branched-chain keto acid oxidation • gene expression • multienzyme complex • BCKA dehydrogenase kinase • metabolic regulation • rats

	INTRODUCTION

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The three branched-chain amino acids (BCAA)³ (leucine, isoleucine and valine) are abundant in tissue proteins. The conditions that increase the catabolism of these essential amino acids result in the loss of body proteins. Among these conditions is starvation. Studies in humans and experimental animals, such as rats, have shown that starvation increases the whole body oxidation of BCAA (1 ,2) .

BCAA are transaminated to branched-chain keto acids (BCKA) before they are oxidized. The key enzyme regulating this oxidation is BCKA dehydrogenase. BCKA dehydrogenase exists in interconvertible phosphorylated (inactive) and dephosphorylated (active) forms. These interconversions are catalyzed by BCKA dehydrogenase kinase (BCKAD kinase), which has been cloned (3) , and by a phosphatase that has not yet been cloned. The BCKAD kinase is tightly linked to BCKA dehydrogenase in the mitochondria and together form a multienzyme complex. Although there have been studies of BCKA dehydrogenase activity in starvation, as yet there has been no study of its protein or mRNA expressions in this condition.

The studies of BCKA dehydrogenase activity in starvation have resulted in conflicting results. For example, Paul and Adibi (4) reported an increase in the hepatic activity in starvation, whereas Gillim et al. (5) did not find any increase in the hepatic activity. A similar disagreement also exists regarding the activity in the skeletal muscle (6 ,7) . These conflicting results and, more importantly, the absence of any knowledge on molecular expression of BCKA dehydrogenase in starvation necessitated the present study. This study focused on the liver and the skeletal muscle because they are the main sources of BCKA dehydrogenase in the body.

	MATERIALS AND METHODS

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Animals and animal treatment.

Male Sprague-Dawley rats (Harlen-Sprague, Indianapolis, IN), weighing 200–250 g were housed individually in temperature-controlled quarters (22°C) with controlled 12-h light/dark cycles. All rats consumed nonpurified diet (LabDiet 5P00 Prolab RMH 3000; PMI Nutritional International, Richmond, IN) and drinking water ad libitum before experimentation. Rats were divided into two groups of 12. The first group served as the control and had free access to food and water. Food was withdrawn from the second group 48 h before killing, but they had free access to drinking water. Between 0900 and 1000 h, rats were anesthetized with halothane, and liver and gastrocnemius muscle were quickly freeze-clamped with precooled Wollenberger clamps in liquid nitrogen. Tissues were stored at -80°C until processing. All of the above procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Isolation and assay of BCKA dehydrogenase.

BCKA dehydrogenase was extracted from freeze-clamped skeletal muscle (8) and liver (9) by polyethylene glycol precipitation. Protein concentration was determined using the Bio-Rad (Richmond, CA) protein assay with bovine serum albumin serving as the standard.

Liver BCKA dehydrogenase activity was assayed spectrophotometrically by measuring the reduction of NAD⁺ (10) . Complete assay mixture contained 30 mmol potassium phosphate buffer/L (pH 7.4), 3 mmol NAD⁺/L, 0.4 mmol CoASH/L, 0.4 mmol thiamine pyrophosphate/L, 2 mmol dithiothreitol (DTT)/L, 5 mmol MgCl₂/L, 10 U pig heart dihydrolipoyl dehydrogenase, 0.1% (v/v) Triton X-100, 0.5 mmol -ketoisovalerate/L and BCKA dehydrogenase sample (1–2 mg of protein) in a final volume of 1.5 mL. All assays were preformed at 30°C, and the enzyme activity is expressed in nmol of NADH formed/(min · g wet starting tissue). Basal BCKA dehydrogenase activity was determined by adding freshly extracted complex directly to the assay mixture. Total BCKA dehydrogenase activity was determined by preincubating an aliquot of the extracted complex with 15 mmol MgSO₄/L, 0.1 mmol -chloroisocaproate/L and 25 mU protein/mg of a broad-specificity phosphoprotein phosphatase (5 ,11) at 37°C. After 30 min of preincubation, an aliquot was removed, and BCKA dehydrogenase activity was determined. All assays were performed in triplicate, and percent active complex was calculated from the ratio of basal to total activities in the same tissue sample.

Due to the low activity of BCKA dehydrogenase in the skeletal muscle, activity in this tissue was assayed by a different method. The activity was determined by measuring the release of ¹⁴CO₂ from -keto-[1-¹⁴C]isocaproate (Amersham, Arlington Heights, IL) (12) . Complete assay mixture contained 25 mmol HEPES, 2 mmol NAD⁺, 0.5 mmol CoASH, 0.5 mmol thiamine pyrophosphate/L, 0.2 mmol Na₂-EDTA, 2 g Brij 58, 1 mmol DTT, 2 mmol MgSO₄, 1 mmol N-2-p-tosyl-L-lysine chloromethyl ketone, 20 mg leupeptin and 0.1 mmol -keto-[1-¹⁴C]isocaproate (2500 dpm/nmol) per L and appropriate amounts of BCKA dehydrogenase complex (0.5–1.5 mg protein) in a final volume of 0.35 mL. All assays were performed in triplicate at 37°C and carried out for 15 min. At the end of this period, the reactions were stopped with 2.5 mol H₂SO₄/L, ¹⁴CO₂ was collected in hydroxide of Hyamine and radioactivity was determined by liquid scintillation spectrometry (12) . Enzyme activity is expressed in nmol/CO₂ released/(min · mg of protein). Total activity was determined using the same preincubation method used for liver. At the end of the preincubation, aliquots were removed and added to the radiochemical assay for BCKA dehydrogenase activity determination.

Rate of inactivation of BCKA dehydrogenase.

BCKA dehydrogenase complex with bound kinase was extracted from freeze-clamped skeletal muscle by the same polyethylene glycol precipitation method used for the isolation of BCKA dehydrogenase from skeletal muscle (8) . BCKA dehydrogenase complex with bound kinase was extracted from freeze-clamped liver as previously described (13 ,14) . The rate of inactivation of BCKA dehydrogenase in the liver and skeletal muscle was determined as described previously (14) . Briefly, the complete reaction mixture contained, in a final volume of 0.2 mL, 30 mmol HEPES/L (pH 7.35), 1.5 mmol MgCl₂/L, 5 mmol DTT/L, 0.1 mmol EDTA/L, 0.5 g Triton X-100/L, 0.1 µmol leupeptin/L, 10 mg trypsin inhibitor/L, 0.5 mmol ATP/L and 0.10 mg of extracted BCKA dehydrogenase complex. Reactions were incubated at 30°C (liver) or 37°C (skeletal muscle) for 10 min. At various time intervals (0–10 min), aliquots (20 µL) were removed and transferred into the appropriate BCKA dehydrogenase assay mixture, and dehydrogenase activity was measured as described previously.

Western blot analysis.

BCKA dehydrogenase and BCKAD kinase were extracted from liver and skeletal muscle by the methods mentioned above. Equal amounts of protein (100 µg) from fed and 48-h starved tissues were suspended in SDS buffer [40 g SDS/L, 0.125 mol Tris · HCl/L (pH 6.8), 20% (v/v) glycerol, 10% ß-mercaptoethanol, 5 g bromophenol blue/L] and boiled for 90 s. Sample were subjected to SDS–10% PAGE in a Laemmli system (15) . Resolved proteins were transferred onto nitrocellulose membranes and subjected to immunoblot analysis. The membranes were incubated with polyclonal antibody raised against either purified BCKA dehydrogenase complex (1:2000) or purified BCKAD kinase (1:500). The membranes were then washed and incubated with the second antibody, peroxidase-conjugated goat anti-rabbit IgG (1:2000 for BCKA dehydrogenase and 1:1000 for BCKAD kinase), as described previously (10 ,14) . Subunits of BCKA dehydrogenase and BCKAD kinase were detected with the ECL Western blotting system of Amersham. The intensity of the bands was quantified by densitometry using Image PC (Scion Corporation, Frederick, MD). Preliminary studies showed linearity of Western blot assays from 50 to 200 µg of protein for E₁, E₁ß and E₂ subunits and from 75 to 225 µg of protein for BCKAD kinase. The correlation coefficients between the amount of protein and ECL image intensity were 0.93, 0.97 and 0.95 for the E₁, E₁ß and E₂ subunits and 0.97 for BCKAD kinase (all P < 0.01).

RNA extraction and Northern blot analysis.

Total cellular RNA from freeze-clamped liver and skeletal muscle of fed and 24- and 48-h starved rats was extracted by RNAzol method (Tel-Test, Friendswood, TX). RNA (20 µg for BCKA dehydrogenase and 25 µg for BCKAD kinase) was fractionated on 9 g agarose/L gels containing formaldehyde and blotted onto a Nytran membrane (Schleider & Schuell, Dassel, Germany). The membranes were hybridized as described previously (16) . Cloned cDNAs encoding the E₁, E₁ß and E₂ subunits of rat BCKA dehydrogenase and BCKAD kinase were kindly provided by Dr. Robert Harris, Indiana University School of Medicine. ³²P-labeled cDNA probes were made by random primer technique (17) ([³²P]dCTP; Du Pont New England Nuclear, Boston, MA; kit for radiolabeling DNA; Pharmacia Biotech, Piscataway, NJ). Blots were subjected to autoradiography with Kodak Biomax MS film at -70°C for 72 h. The intensity of bands was quantified by densitometry using Image PC software (Scion, Frederick, MD). RNA level for each sample was normalized to the abundance of ß-actin RNA (cDNA clone; Clontech Laboratories, Palo Alto, CA), which served as an internal control for minor variations in sample loading.

Statistical analysis.

All data are presented as means ± SEM, n = 4–6 six rats per group. Student’s t test was used for statistical analysis of the data.

	RESULTS

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BCKA dehydrogenase activity.

The BCKA dehydrogenase activity was 100% greater in the liver [73.5 ± 8.5 versus 44.86 ± 2.9 nmol/(min · mg protein), P < 0.05] and gastrocnemius muscle [0.33 ± 0.03 versus 0.17 ± 0.02 nmol/(min · mg protein), P < 0.01] of starved than fed rats, establishing that starvation increased BCKA dehydrogenase activities in both tissues.

Mechanisms of increased BCKA dehydrogenase activity.

There are two mechanism for increasing the level of activity of BCKA dehydrogenase: one is to increase its activity state, and the other is to increase its protein mass. Both of these mechanisms were investigated in the present experiment.

The activity state was greater by 3-fold in the skeletal muscle (11 ± 2% versus 3 ± 1%, P < 0.05) of starved rats. In contrast, there was no significant difference between the activity states in the liver of starved and fed rats (98.2 ± 2% versus 93 ± 1%).

To investigate the protein expression of BCKA dehydrogenase, we determined the protein expression of its individual subunits. BCKA dehydrogenase is composed of three catalytic proteins, designated E₁, E₂ and E₃. The E₁ component is further composed of (E₁) and ß (E₁ß) subunits. The E₁ and E₂ components are specific for BCKA dehydrogenase, whereas the E₃ is common to other dehydrogenases. Therefore, the present study included the investigation of the protein expression of the E₁, E₁ß and E₂ subunits.

Western blot analysis of BCKA dehydrogenase subunits in the liver showed no significant difference between the expressions of E₂ or E₁ß but a significantly greater protein expression of E₁ in starved than in fed rats (Fig. 1 ). Western blot analysis of BCKA dehydrogenase subunits in the skeletal muscle showed no significant difference between the expressions of either E₂ or E₁ but significantly lower protein expression of E₁ß in starved than in fed rats (Fig. 2 ).

View larger version (38K): [in this window] [in a new window]   Figure 1. Protein expression of branched-chain keto acid dehydrogenase subunits in liver of fed and 48-h starved rats. Top, Western blot of a representative sample from fed and starved rats. Molecular mass markers are shown on the right. Bottom, Quantitative densitometric analysis of Western blots of branched-chain keto acid dehydrogenase subunits. Values are given as means ± SEM, n = 4–6 rats; expressed as percentage of fed rats. *P < 0.05 versus fed rats.

View larger version (51K): [in this window] [in a new window]   Figure 2. Protein expression of branched-chain keto acid dehydrogenase subunits in skeletal muscle. Top, Western blot of a representative sample from fed and 48-h starved animals. Molecular mass markers are shown on the right. Bottom, Quantitative densitometric analysis of Western blots of branched-chain keto acid dehydrogenase subunits. Values are given as means ± SEM, n = 4–6 rats; expressed as percentage of fed rats. *P < 0.05 versus fed rats.

Inactivation of BCKA dehydrogenase.

Inactivation of BCKA dehydrogenase is a key process for establishing the activity state of this enzyme. Therefore, we determined the rate of inactivation of BCKA dehydrogenase in tissues of fed and starved rats. The rate of inactivation was studied by determining BCKA dehydrogenase activity as a function of time when ATP was added to the extracted enzyme complex (4 ,5) . The rate of inactivation was calculated as the first-order kinetic constant, k/min, of semilog plots of residual BCKA dehydrogenase activity versus time (Fig. 3 ). The rate of inactivation was significantly (P < 0.01) faster in the skeletal muscle of fed than starved rats (0.11 ± 0.02 versus 0.06 ± 0.02), whereas it was not significantly different in the liver (0.20 ± 0.03 versus 0.17 ± 0.04).

View larger version (13K): [in this window] [in a new window]   Figure 3. Inactivation of branched-chain keto acid dehydrogenase in skeletal muscle (A) and liver (B). Plot of percent activity remaining during ATP-dependent inactivation of dehydrogenase activity as a function of time. Insets: Semilog plot of rate of loss of dehydrogenase activity versus time. Slopes correspond to rates of inactivation of branched-chain keto acid dehydrogenase (k/min). Values are given as means, n = 4–6 rats; expressed as percentage of fed rats.

The rate of inactivation of BCKA dehydrogenase is mediated by BCKAD kinase. We, therefore, determined the protein and mRNA expressions of the gene encoding this enzyme in the liver and skeletal muscle of fed and starved rats. We found no significant differences in any of these variables (results not shown).

	DISCUSSION

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Starvation increases the concentrations of BCKA in plasma and tissues (2 ,18) . These increases are consequences of increased concentrations of BCAA (19) and their transamination (20) in starvation. Although the metabolic alteration of BCAA oxidation has been described in starvation (21) , this is the first study of the effect of starvation on protein and mRNA expressions of BCKA dehydrogenase subunit genes. Increases in the protein expression of the individual subunits were not responsible for the increased BCKA dehydrogenase activity in the skeletal muscle. In fact, starvation significantly decreased the protein expression of E₁ß in the skeletal muscle. The importance of the decrease in E₁ß expression is unclear, because this decrease did not prevent starvation from increasing BCKA dehydrogenase activity in the skeletal muscle. On the other hand, the increase in BCKA dehydrogenase activity in the liver of the starved rat was accompanied by an increase in protein expression of E₁. It has been proposed that E₁ may serve as an activator of BCKA dehydrogenase in the liver (22 ,23) . Therefore, the increased expression of E₁ may be involved in the increased hepatic BCKA dehydrogenase activity. Alternatively, the E₁ constituent of the BCKA dehydrogenase complex may be limiting, and the increase in this subunit may increase the catalytic activity of the enzyme. Regardless of the explanation, the mechanisms of these alterations in protein expressions appeared to be posttranscriptional, because the mRNA expressions of the genes encoding E₁ and E₁ß subunits did not change in starvation. The posttranscriptional changes were selective, and included both up- and down-regulation (Figs. 1 and 2) .

The present results support our previous suggestion that the muscle is largely responsible for increased oxidation of BCKA in starvation (24) . After 48 h of starvation, there is a substantial decrease in the weight of the liver, whereas there is very little decrease in the weight of the gastrocnemius muscle (19) . Consequently, if the BCKA dehydrogenase activity, determined in the present experiment, is expressed per whole weight of organ, there is no difference between the activities in the liver, whereas the activity is still 100% greater in the skeletal muscle of starved than fed rats. Furthermore, among the molecular and biochemical differences observed in the two tissues, the most striking one was the decreased rate of inactivation of BCKA dehydrogenase in the skeletal muscle of starved rats (Fig. 3) .

The factors affecting the above rate of inactivation include the concentrations of BCKAD kinase, -ketoisocaproate (25) and ATP (4) . Because our study showed that there was no decrease in the protein expression of the kinase, cellular factors, such as those mentioned above, appear to be responsible for the decreased rate of inactivation of BCKA dehydrogenase in starved rats. Indeed, starvation increases the concentration of -ketoisocaproate (2 ,18) and decreases the concentration of ATP (4) , both of which favor a decrease in the rate of inactivation. However, because of the addition of -ketoisocaproate and ATP to the assay mixture, whether any of these factors played a role cannot be ascertained. Clearly, a problem worthy of further investigation is the identity of the factors regulating the inactivation of BCKA dehydrogenase in starvation.

The molecular biology of BCKA dehydrogenase has been studied in conditions such as changes in dietary protein intake (26) , clofibrate feeding (14) , diabetes (27 ,28) and exercise (29) . Among these conditions, the one with the most relevance to the present study is diabetes, because diabetes is a form of tissue starvation. However, comparison of the results of studies indicates that there are distinct differences between starvation and diabetes in the responses of the liver and the skeletal muscle to these metabolic alterations. For example, in diabetes there were increases in the protein expressions of E₁, E₁ß and E₂ and decreases in the protein expressions of BCKAD kinase in both the liver and skeletal muscle (27 ,28) . Except for the increase in E₁ in the liver, these changes were not observed in starvation. These differences indicate a) complex regulation of expressions of BCKA dehydrogenase in metabolic alterations and b) different patterns of regulation of the activity of this enzyme under differing metabolic alterations.

Up-regulation of BCKA dehydrogenase serves important functions in starvation. For example, it prevents very great increases in BCKA concentrations. High concentrations of BCKA are neurotoxic. Because of the increased BCKA production in starvation, if the above enzymatic alteration had not occurred, it is very likely that the BCKA concentrations would have been much higher than observed (2 ,18) .

In conclusion, by revisiting a condition that awakened the interest in metabolic regulation of BCKA dehydrogenase, the present study brings new evidence on the importance of cellular versus molecular mechanisms of this regulation. It shows that in starvation, the primary mechanism of regulation is at the cellular, and not at the molecular, level. The present results, together with our previous results (14 ,27 ,28) , establish a spectrum of cellular and molecular mechanisms for regulation of BCKA dehydrogenase in metabolic alterations.

	FOOTNOTES

 
¹ Supported in part by National Institutes of Health grant DK15855.

³ Abbreviations used: BCAA, branched-chain amino acids; BCKA, branched-chain keto acid; BCKAD kinase, branched-chain keto acid dehydrogenase kinase; DTT, dithiothreitol.

Manuscript received January 10, 2001. Initial review completed February 5, 2001. Revision accepted March 12, 2001.

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