Regulation of branched-chain alpha-keto acid dehydrogenase kinase expression in rat liver

Branched-chain amino acids are toxic in excess but have to be conserved for protein synthesis. This is accomplished in large part by control of the activity of the branched-chain alpha-keto acid dehydrogenase complex by phosphorylation/dephosphorylation. Regulation of the activity of the hepatic enzyme appears particularly important, at least in rats, since an exceptional high activity of the complex in this tissue makes the liver the primary clearing house for excess branched-chain alpha-keto acids released by other tissues. The degree to which the branched-chain alpha-keto acid dehydrogenase complex is inactivated by phosphorylation is determined by the activity of the branched-chain alpha-keto acid dehydrogenase kinase, which is itself regulated by allosteric effectors as well as factors that affect its level of expression. Well established among these are the alpha-keto acid produced by leucine transamination, which is a potent inhibitor of the kinase, and starvation for dietary protein, which causes increased expression of the branched-chain alpha-keto acid dehydrogenase kinase. The latter finding resulted in the working hypothesis that nutrients and hormones regulate expression of the branched-chain alpha-keto acid dehydrogenase kinase. Evidence has been obtained for the involvement of thyroid hormone, glucocorticoids and ligands for peroxisome proliferator-activated receptor alpha. Thyroid hormone induces, whereas glucocorticoids and peroxisome proliferator-activated receptor alpha ligands repress, expression of the kinase. Increased blood levels of thyroid hormone are proposed to be responsible for increased expression of branched-chain alpha-keto acid dehydrogenase kinase in animals starved for protein.     

Activation of liver branched-chain alpha-keto acid dehydrogenase in rats by excesses of dietary amino acids

As part of an effort to explain the leucine-induced depressions of plasma isoleucine and valine concentrations, and the concomitant stimulation of valine oxidation in vivo, branched-chain alpha-keto acid dehydrogenase (BCKAD) activity was measured in livers from rats that were fed for only 6 h/d large quantities of individual amino acids in a low protein diet. Preincubation of homogenates with buffer containing Mg2+ and Ca2+ allowed estimation of fully active complex. Cytosolic and mitochondrial branched-chain-amino-acid aminotransferase (BCAAT) activities were also measured in livers of rats fed an excess of leucine. The percentage of BCKAD in the active form in livers of rats fed the low protein diet containing an excess of leucine, isoleucine, valine or phenylalanine for 2 d was double that of rats fed the low protein control diet (control, leucine, isoleucine, valine and phenylalanine groups having, respectively, 45 +/- 2, 85 +/- 7, 85 +/- 3, 95 +/- 5, and 81 +/- 4% of hepatic BCKAD in the active form). Consumption of a low protein diet containing an excess of leucine had no significant effect on either cytosolic or mitochondrial BCAAT activities of liver. The response of BCKAD in liver can contribute to the leucine-induced stimulation of valine oxidation in vivo but analysis of the results of this study leads to the conclusion that other mechanisms, probably in nonhepatic tissues, must also be involved.     

Regulation of branched-chain amino acid catabolism: nutritional and hormonal regulation of activity and expression of the branched-chain alpha-keto acid dehydrogenase kinase

Branched-chain alpha-keto acid dehydrogenase kinase is responsible for the inactivation and phosphorylation of the branched-chain alpha-keto acid dehydrogenase complex, the enzyme that catalyses the committed step of branched-chain amino acid catabolism. The activity of the branched-chain alpha-keto acid dehydrogenase complex is inversely correlated with kinase activity, suggesting that the relative activity of the kinase is the primary regulator of the activity of the complex. It has been shown that kinase activity and expression are affected by nutritional states imposed by low-protein diet feeding, starvation, diabetes, and exercise. Evidence has also been presented that certain hormones, particularly insulin, glucocorticoid, thyroid hormone and female sex hormones, affect the activity and expression of the kinase. The findings indicate that nutritional and hormonal control of the activity and expression of branched-chain alpha-keto acid dehydrogenase kinase provides an important means of control of the activity of the branched-chain alpha-keto acid dehydrogenase complex, with inactivation serving to conserve branched-chain amino acids for protein synthesis in some situations and activation serving to provide carbon for gluconeogenesis in others.     

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.     

High branched-chain alpha-keto acid intake, branched-chain alpha-keto acid dehydrogenase activity, and plasma and brain amino acid and plasma keto acid concentrations in rats

Diets containing high quantities of individual branched-chain alpha-keto acids (BCKAs) or a combination of BCKAs as used for treatment of renal disease were fed to rats. When the diet contained a single BCKA, its concentration was high in plasma and the concentration of its corresponding amino acid was high in plasma and brain. Liver BCKA dehydrogenase (BCKD) was 42% active in control rats. Consumption of diets containing 0.38 mol/kg diet of alpha-ketoisocaproate (KIC), alpha-keto-beta-methylvalerate (KMV), or alpha-ketoisovalerate (KIV) resulted in complete activation of liver BCKD. Consumption of the diet containing the combination of BCKAs increased basal BCKD activity of liver twofold. Muscle BCKD was activated after feeding the KIV diet (2-fold), the KIC diet (3-fold), and the KMV diet (15-fold). Total BCKD activity of liver and muscle was unaffected by dietary treatments. Activation of liver and muscle BCKD by dietary BCKA is consistent with their ability to inhibit BCKD kinase in vitro.     

Effect of branched-chain keto-acids and dietary protein content on the activity of branched-chain amino acid transferase in rat tissues

Rats fed branched-chain keto-acids in place of branched-chain amino acids exhibited increased specific activity of branched-chain amino acid transferase (BATase) in muscle, intestine, brain and liver as compared with controls fed sufficient diet to achieve comparable weight gain. This increase was observed whether or not methionine and phenylalanine were also replaced by their N-free analogues. Kidney BATase was unaffected. Rats fed a protein-free diet exhibited higher BATase specific activity in kidney, brain, liver and intestine than rats fed diets containing 6% casein; but little change in specific activity in these organs was seen as casein intake was progressively increased from 6% to 18%. Muscle BATase specific activity was the same between 0 and 18% dietary casein. The results show that branched-chain keto-analogues augment BATase in several tissues, including muscle. In contrast, varying casein intake from 6% to 18% had little effect, although protein-feeding augments BATase in some organs.     

Hepatic branched-chain alpha-keto acid dehydrogenase complex in female rats: activation by exercise and starvation.

The effects of acute exercise and starvation on hepatic branched-chain alpha-keto acid dehydrogenase (BCKDH) complex activity were examined in female rats fed high (30%)- or low (8%)-protein diets. The total activity of the complex was significantly higher in the high protein-fed rats than in the low protein-fed rats but was not affected by acute exercise and starvation in either diet group. The proportion of the active form of BCKDH complex was less than 10% in both diet groups. Acute exercise and starvation markedly increased the active form of the complex in both diet groups. The activity of BCKDH kinase, which is responsible for inactivation of the BCKDH complex by phosphorylation, tended to be decreased by acute exercise and starvation in both diet groups. These results suggest that the activity of the BCKDH kinase is an important factor determining the proportion of the active form of BCKDH complex in exercise and starvation, and that the female rat is a useful model for studying the regulation of hepatic BCKDH complex activity.     

Effects on plasma amino acid concentrations and hepatic branched-chain alpha-keto acid dehydrogenase activity of feeding rats diets containing 9 or 50% casein

Plasma concentrations of amino acids and alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate decarboxylation activities in livers of rats trained to eat 9 or 50% casein diets for 5 hours/day, were measured one-half hour before and one-half and 3 hours after the start of the feeding period. Decarboxylation of both alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate by liver increased significantly within one-half hour after rats had ingested either the 9 or the 50% casein diet. Liver decarboxylation activity of rats fed the 50% casein diet was from two- to fivefold higher than that of rats fed the 9% casein diet. The greatest difference was observed when calcium, NAD and coenzyme A were included in the decarboxylation assay medium. Although the activity of the branched-chain alpha-keto acid dehydrogenase increased in response to food ingestion, plasma concentrations of branched-chain amino acids also increased greatly after the ingestion of food. The similarity in the responses of alpha-ketoisocaproate and alpha-keto-gamma- methiolbutyrate decarboxylation in rats fed diets differing in protein content and subjected to different feeding regimens allows us to suggest that the branched-chain alpha-keto acid dehydrogenase is responsible, in part, for the oxidative decarboxylation of the alpha-keto acid analog of methionine. J. Nutr . 114: 1025-1034, 1984.     

Influence of dietary leucine content on the activities of branched-chain amino acid aminotransferase (EC 2.6.1.42) and branched-chain alpha-keto acid dehydrogenase (EC 1.2.4.4) complex in tissues of preruminant lambs

1. Branched-chain amino acid aminotransferase (EC 2.6.1.42; BCAAT) and branched-chain alpha-keto acid dehydrogenase (EC 1.2.4.4; BCKDH) activities were measured preruminant lamb liver, longissimus dorsi muscle, kidney, jejunum and adipose tissue, 2 h after a meal with or without an excess of leucine. 2. Skeletal muscle contained about 70% of the total basal BCAAT activities of the tissues studied whereas liver contained about 60% of the total BCKDH activities of these tissues. 3. BCAAT activities were very low in preruminant lamb tissues. BCKDH was more phosphorylated in tissues of preruminant lambs than in rats, especially in liver. These low catalytic potentialities might contribute to a low rate of branched-chain amino acid catabolism in sheep. 4. Ingestion of an excess of leucine led to an increase in liver and jejunum BCAAT activities and activation of BCKDH in jejunum.     

High levels of dietary amino and branched-chain alpha-keto acids alter plasma and brain amino acid concentrations in rats

Plasma and brain amino acid and plasma branched-chain alpha-keto acid (BCKA) concentrations were measured in rats fed diets containing high levels of individual amino and alpha-keto acids. Consumption of a low-protein (9% casein) diet high in leucine or alpha-ketoisocaproate depressed plasma concentrations of isoleucine and valine and their respective keto acids, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate. High dietary levels of alpha-keto-beta-methylvalerate or alpha-ketoisovalerate (but not of isoleucine or valine) depressed plasma concentrations of the other BCKA and their respective branched-chain amino acids (BCAA). Consumption of a low protein, high phenylalanine diet depressed plasma concentrations of both BCAA and BCKA. Brain large neutral amino acid pools of rats fed all low-protein, high-amino acid diets were depleted. Consumption of diets high in individual BCKA increased brain concentrations of aromatic amino acids. In this study of rats allowed to feed for only 6 h/d, elevated brain phenylalanine concentration was associated with a significant depression of food intake, whereas elevated brain BCAA concentrations were not. Also, elevated plasma BCKA concentrations, comparable with those observed in maple syrup urine disease, were accompanied by elevations in concentrations of aromatic amino acids in brain but not in plasma.     

Lowered concentrations of branched-chain amino acids result in impaired growth and neurological problems: insights from a branched-chain alpha-keto acid dehydrogenase complex kinase-deficient mouse model

Excess circulating levels of branched-chain amino acids (BCAA), as seen in maple syrup urine disease, result in severe neuropathology. A new mouse model, deficient in the kinase that controls BCAA catabolism, shows that very low circulating levels of BCAA are also associated with neuropathology, including the development of epileptic seizures. These mice clearly demonstrate the need to control essential amino acid levels within both upper and lower limits.     

Regulation of protein synthesis by branched-chain amino acids

Historically, amino acids have been viewed as precursors for protein synthesis as well as metabolic substrates. Recently, a new role for amino acids as regulators of mRNA translation has been identified. In this role, they modulate the phosphorylation state of proteins that represent important control points in translation initiation, including the translational repressor 4E-BP1 and the ribosomal protein S6 kinase S6K1. When administered orally to fasted rats the branched-chain amino acids are particularly effective in stimulating translation initiation. Of the branched-chain amino acids, leucine is most potent. Interestingly, leucine administration stimulates global rates of protein synthesis in skeletal muscle but not in liver. However, in liver, branched-chain amino acids enhance the translation of a particular set of mRNAs typified by those encoding the ribosomal proteins and translation elongation factors, suggesting that branched-chain amino acids upregulate the capacity of the tissue to synthesize protein.     

Effects of dietary linoelaidate on fatty acid composition and phospholipase activity in rat liver microsomes

The activity of calcium-stimulated microsomal phospholipase towards endogenous membrane bound phospholipids was monitored by analyzing the fatty acids released using gas chromatography. Activation by calcium (10 mM) resulted in the release of 2.1 nmoles fatty acid/10 mg microsomal protein/hr. The pattern of release of particular fatty acids reflected their concentration in the microsomal phospholipids.The effects of feeding 10 and 50% dietary linoelaidate (t,t18:2) and hydrogenated tallow (i.e., essential fatty acid deficient) on microsomal fatty acids and phospholipase activity was determined. The levels of t,t18:2 in the microsomal phospholipids increased with dietary intake and duration of feeding and attained 8.4% of total fatty acids after 11 weeks. There was a concomitant increase in linoleic acid and a decrease in arachidonic acid levels in microsomal lipids. The microsomal phospholipids of animals receiving exclusively hydrogenated tallow contained only trace amounts of linoleic and arachidonic acid after four weeks. The amount of eicosatrienoic acid increased dramatically in these animals. Palmitoleic and oleic acid also increased. The calcium-stimulated phospholipase activity in liver microsomes was unchanged by alterations in the fatty acid composition of the phospholipids.     

Regulation by dietary fats of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase in rat liver

The effects of various dietary fats on the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in rat liver microsomes, the rate-limiting enzyme in cholesterogenesis, were examined. A series of experiments demonstrated the dependency of the HMG-CoA reductase activity on the nature of dietary fats. When saturated fats with chain length of 12 to 18 were the dietary sources and were fed at the 10% level for 19 days, feeding fats with shorter chain fatty acids caused a lower enzyme activity compared to those with longer chain fatty acids. The activity was also regulated by the degree of unsaturation of dietary fats; when safflower oil, camellia oil or tristearin were fed at the 10% level for 18 days, the higher the unsaturation, the lower the activity. When trimyristin or tripalmitin were fed at the 10% level for 14 days, addition of essential fatty acid, at the level of minimum daily requirement (1% was replaced by safflower oil), did not affect the enzyme activity. Through the rate of incorporation of mevalonate into cholesterol in the 12,500 x g supernatant fraction of the liver was also found to be influenced by the types of dietary fats, the extent of the response appeared much smaller than that of HMG-CoA reductase. No consistent correlation between the HMG-CoA reductase activity and the content of microsomal cholesterol or cholesteryl ester and the fatty acid composition of microsomal lipids was observed.     

Activities of branched-chain amino acid--degrading enzymes in liver from rats fed different dietary levels of protein

The relationships among dietary protein intake, plasma branched-chain amino acid (BCAA) and keto acid (BCKA) concentrations, and liver BCAA-degrading enzyme activities were investigated in rats fed, for 5 h/d for 2, 6 or 9 d, diets containing from 0 to 50% casein. Plasma, liver and muscle BCAA concentrations were proportional to protein intake over the entire range tested; plasma BCKA concentration, however, was proportional only in the range from 0 to 20% casein, after which a plateau was reached. By d 2, liver cytosolic BCAA aminotransferase activity had increased in rats fed 50% casein; by d 9, activity had increased in rats fed 0 or 5% casein as well. Liver mitochondrial BCAA aminotransferase activity was unresponsive to dietary treatment. Basal liver BCKA dehydrogenase activity and the percent active complex were proportional to protein intake on d 2 and 6. On d 2, total BCKA dehydrogenase activity was the same in all groups; by d 6, total activity had increased in rats fed 30 or 50% casein. We conclude that although the adaptive changes in BCAA-degrading enzyme activities are small, they are sufficient to compensate for excessively high or low protein intakes, so that tolerable concentrations of BCAA and BCKA are maintained.     

Role of dietary protein in rat pancreatic enzyme secretory response to a meal

The role of dietary protein in the pancreatic enzyme secretory response to complete test meals was studied in rats fitted with chronic bile-pancreatic fistulas. Pancreatic enzyme secretory response was observed by collecting combined bile-pancreatic juice and continuously returning it to the intestine by a stream-splitter, which diverted 5% for assay. All rats were fed ad libitum a 24% casein diet during the recovery period. Four days postoperative, rats were infused intragastrically with 5 g of isonitrogenous, complete test meals containing casein (24%), alpha-protein (soy protein), amino acids patterned after casein or alpha-protein, casein + lima bean trypsin inhibitor (LBTI), and a nitrogen-free diet. Pancreatic enzyme secretion under basal conditions and in response to the test meals was determined. The pancreatic response to test meals containing intact casein as nitrogen source was the same as the response to nitrogen-free test meals, and was only slightly greater (not statistically significantly) than the response to test meals containing amino acids. Test meals containing alpha-protein, which was devoid of soybean trypsin inhibitor activity, were much more potent than corresponding amino acid test meals or nitrogen-free test meals. Addition of LBTI greatly increased the pancreatic response to casein test meals. The results indicate that intragastric infusion of diets containing casein as nitrogen source do not stimulate greater pancreatic enzyme secretion than test meals of nitrogen-free or amino acid diets under normal physiological conditions, and it is proposed that this is a consequence of the large reserve capacity of the exocrine pancreas.     

Sex-dependent developmental change of rat liver cytosolic alcohol dehydrogenase activity

Rat liver cytosolic alcohol dehydrogenase (ADH) is the principal enzyme which catalyzes the oxidation of ethanol. ADH activity is known to be significantly higher in females than in males. However, the precise mechanism of the sex-difference in ADH activity is uncertain. Recently, we have shown that the inhibitory action of androgen and the slight facilitatory actions of progestin and estrogen are involved in the mechanism of sex-difference in adult rat liver ADH activity. In the present study, we studied the difference of the postnatal developmental changes of ADH activity between males and females. ADH activity increased rapidly after birth up to about 12 d of age, and was not different in the two sexes. In female rats, ADH activity peaked at about 30 d of age and then increased gradually to plateau levels. However, the ADH activity of male rats fell markedly between 30 and 45 d old, and the reduced enzyme activity was observed until 104 d old. In conclusion, the ADH activity of male rats fell between 30 and 45 d after birth. At the ages over the turning point, the ADH activity was significantly lower in male than in female rats.