Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

The effect of starvation on branched-chain 2-oxo acid oxidation in rat muscle.

Oxidative-decarboxylation rates of branched-chain amino acids in rat hemidiaphragm and of branched-chain 2-oxo acids in hemidiaphragm, soleus muscle and heart slices of 110-120 g rats were increased considerably by 3-4 days of starvation, when they were calculated from the specific radioactivity in the medium. When the supply from endogenous protein degradation to the oxidation-precursor pool was severely limited by transaminase inhibitors, oxidative-decarboxylation rates of branched-chain 2-oxo acids rose significantly. Since this apparent increase was relatively larger in preparations from fed rats than from 3-days-starved rats, the differences in oxidation rates with nutritional state became less or even not significant. With rat heart the smaller dilution of the oxidation precursor pool after starvation is in accordance with the reported decrease in protein breakdown. Since protein degradation increases with starvation in skeletal muscles, we suggest that the amino acid pool arising from protein degradation is more segregated from the oxidation precursor pool in muscles from starved than from fed rats. We conclude that starvation increases branched-chain amino acid and 2-oxo acid oxidation in skeletal and cardiac muscle considerably less than has been suggested by previous studies.     

Effect of starvation and exercise on actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Starvation does not change the actual activity per g of tissue of the branched-chain 2-oxo acid dehydrogenase in skeletal muscles, but affects the total activity to a different extent, depending on the muscle type. The activity state (proportion of the enzyme present in the active state) does not change in diaphragm and decreases in quadriceps muscle. Liver and kidney show an increase of both activities, without a change of the activity state. In heart and brain no changes were observed. Related to organ wet weights, the actual activity present in the whole-body muscle mass decreases on starvation, whereas the activities present in liver and kidney do not change, or increase slightly. Exercise (treadmill-running) of untrained rats for 15 and 60 min causes a small increase of the actual activity and the activity state of the branched-chain 2-oxo acid dehydrogenase complex in heart and skeletal muscle. Exercise for 1 h, furthermore, increased the actual and the total activity in liver and kidney, without a change of the activity state. In brain no changes were observed. The actual activity per g of tissue in skeletal muscle was less than 2% of that in liver and kidney, both before and after exercise and starvation. Our data indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and to a smaller extent in kidney and skeletal muscle in fed, starved and exercised rats.     

Effect of fasting, branched-chain amino acids, and glucocorticoids on histone H1 extractability from rat skeletal muscle.

The effect of 72 h fasting, nutritional therapy of fasted rats, and acute and chronic glucocorticoid treatment on the yield of histone H1 from rat hind limb muscles was determined. Fasting significantly enhanced the extractability of muscle H1. The effect of treating starved rats with glucose alone, or with glucose supplemented with branched-chain amino acids (BCAA), or with two commercial preparations of mixtures of essential and non-essential amino acids was evaluated. Treatment of starved rats with glucose alone significantly decreased H1 extractability from muscles, but isocaloric treatment with glucose supplemented with BCAA or two commercial preparations of amino acid mixtures was more effective. Glucocorticoid treatment for 5 days enhanced the yield of H1 from muscles less than starvation. The enhanced H1 extractability from muscles noted in starved rats is similar to that reported in rats with insulinopenic diabetes and may reflect changes in nuclear fragility.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.     

Peptide inhibitors MAP the way towards fighting anthrax pathogenesis

BCAAs (branched-chain amino acids) are indispensable (essential) amino acids that are required for body protein synthesis. Indispensable amino acids cannot be synthesized by the body and must be acquired from the diet. The BCAA leucine provides hormone-like signals to tissues such as skeletal muscle, indicating overall nutrient sufficiency. BCAA metabolism provides an important transport system to move nitrogen throughout the body for the synthesis of dispensable (non-essential) amino acids, including the neurotransmitter glutamate in the central nervous system. BCAA metabolism is tightly regulated to maintain levels high enough to support these important functions, but at the same time excesses are prevented via stimulation of irreversible disposal pathways. It is well known from inborn errors of BCAA metabolism that dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their alpha-keto acid metabolites results in neural dysfunction. In this issue of Biochemical Journal, Joshi and colleagues have disrupted the murine BDK (branched-chain alpha-keto acid dehydrogenase kinase) gene. This enzyme serves as the brake on BCAA catabolism. The impaired growth and neurological abnormalities observed in this animal show conclusively the importance of tight regulation of indispensable amino acid metabolism.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.     

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.     

The esg locus of Myxococcus xanthus encodes the E1α and E1β subunits of a branched-chain keto acid dehydrogenase

The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1α and E1β subunits) of a branched-chain keto acid dehydrogenase (BCKAD) that is involved in branched-chain amino acid (BCAA) metabolism. The properties of an esg ::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild-type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched-chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched-chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg-encoded enzyme is involved in the synthesis of branched-chain fatty acids during vegetative growth, and these compounds are used later in cell-cell signalling during development.     

Evidence for the presence of a threshold weight for entering diapause in the yellow-spotted longicorn beetle, Psacothea hilaris

Under long-day conditions larvae of Psacothea hilaris (Coleoptera: Cerambycidae) pupate after the 4th or 5th instar, while under short-day conditions they undergo 2-4 nonstationary supernumerary molts and eventually enter diapause. To explore the possibility of a threshold weight for entering diapause, P. hilaris larvae were deprived of food on days 0 (day of ecdysis), 4 or 8 of the 4th, 5th and 6th instars under short-day conditions. Within the first 40 days of starvation, 60% of the larvae starved starting on day 0 of the 4th instar died, but all the larvae starved at later stages survived. The incidence of diapause in these survivors was determined by the occurrence of pupation after a temporary chilling at 15 degrees C for 15 days. Diapause incidence increased as the onset of starvation was delayed; from 11% in the larvae starved on day 0 of the 5th instar to 100% in the larvae starved on day 4 and day 8 of the 6th instar. Analysis of the relationship between the initial weight of a respective larva at the onset of starvation and its pupation success revealed that none of the larvae weighing 690 mg did. This finding suggests the presence of a threshold weight (about 600 mg), below which larvae are incapable of entering diapause. We discuss these findings with reference to the life history of P. hilaris.     

Disproportionate reduction of actin synthesis in hearts of starved rats

We examined the synthesis of proteins in rat myocardium after starvation. Rates of total protein synthesis in myofibrillar and nonmyofibrillar fractions of myocardium of starved animals were reduced similarly (to 70-80% of the rates in hearts of fed animals, p less than 0.002), but rates of synthesis of some individual proteins were affected discoordinately. Radiolabeled proteins from atrial and ventricular explants, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that starvation for 2 days reduced the rate of cardiac actin synthesis to 26-38% of control levels, while the rate of myosin heavy chain synthesis in the same hearts was only moderately reduced (74-80% of control levels). This starvation-induced reduction in actin synthesis could be accounted for at least in part by disproportionately decreased levels of actin mRNA in starved hearts, as revealed by Northern blot hybridization and by in vitro translation analysis. The dramatic decrease in cardiac actin synthesis was rapidly reversible, and actin synthesis returned to normal after a single day of refeeding. The selective reduction of actin synthesis after starvation was specific for the heart: rates of myosin heavy chain and actin synthesis in skeletal muscles (soleus and extensor digitorum longus) were coordinately reduced in response to starvation. To our knowledge, this is the first example of such dramatic discoordinate regulation of myofibrillar protein synthesis in response to a physiological stimulus.     

Radioglucose metabolism by richardson's ground squirrels in the weight-gain and weight-loss phases of the circannual cycle

Summary Captive fed, starved, and refed Richardson's ground squirrels in the weight-gain and weight-loss phases of the circannual cycle were injected with radioglucose and the activity of the label in skeletal muscle proteins and white adipose tissue lipids four hours after injection was used to determine if lean body mass and white adipose tissue would be rapidly restored when starved animals were refed. Starvation for six days reduced carcass mass 27–31% and white adipose tissue mass 23–24% (Table 1). Activity of the label in both tissues of weight-gain and weight-loss animals was reduced by starvation. After four days of refeeding activities retured to levels similar to those in fed animals, with the exception of lower activity in skeletal muscle proteins of weight-gain animals. Furthermore, activity in each tissue fraction of starved and refed weight-gain animals was similar to that in weight-loss animals when expressed as per cent of activity in the respective fed state (Table 2). Radioglucose incorporation indicated that when skeletal muscle and adipose tissue are depleted by starvation, distribution of the label upon refeeding is similar to that in the fed state. Four days after refeeding weight-gain phase ground squirrels had restored 5.5 g of lean body mass and 7.5 g of adipose tissue, including 1.4 g (6 kcal) of protein and 7.0 g (66 kcal) of lipid, respectively. These results are also consistent with the fed state, in which weight-gain animals were depositing more lipid than lean body mass.     

Activation of hepatic branched-chain alpha-keto acid dehydrogenase complex by tumor necrosis factor-alpha in rats

Tumor necrosis factor-alpha (TNFalpha) promotes oxidation of branched-chain amino acids (BCAA). BCAA catabolism is regulated by branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which is regulated by phosphorylation-dephosphorylation of the E1alpha subunit at Ser293. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined the effects of TNFalpha administration on hepatic BCKDH complex and kinase in rats. Rats were intravenously administered with 25 or 50 microg TNFalpha/kg body weight 4 h prior to sacrifice. The TNFalpha treatment at both doses elevated the activity state (percentage of the active form) of BCKDH complex from 22% to 69% and 86%, respectively, and the amount of phospho-Ser293 on the E1alpha subunit in each group of rats corresponded inversely to the activity state of BCKDH complex. The TNFalpha treatment of rats significantly decreased the activity as well as the bound form of BCKDH kinase. These results suggest that the decrease in the bound form of kinase is involved in the mechanism responsible for TNFalpha-induced activation of the BCKDH complex.     

Degradation of cell constituents during starvation of Salmonella enteritidis

Salmonella enteritidis starved in Fernbach flasks used acid-alcohol-soluble material and RNA as endogenous reserves during starvation. Organisms starved in a fermentor system consumed acid-alcohol-soluble material, RNA and protein to maintain viability. Half-life survival times were 132 h and 118 h for the Fernbach and fermentor-starved cells, respectively. The acid-alcohol-soluble fraction of the cell consisted mainly of peptides or protein. This fraction accounted for most of the loss of label from 14C-labelled cells during the first 5 days of starvation and presumably contains the primary endogenous reserve. Although the residue fraction of fermentor-starved cells provided 35% of the total loss of 14C from the cells by the 5th day, a small net increase in 14C activity of the residue fraction of Fernbach-starved cells was observed. Differences observed appeared to be due to the method of starvation.