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Uteroplacental insufficiency causes intrauterine growth restriction (IUGR) and decreases plasma levels of the branched-chain amino acids in both humans and rats. Increased fetal oxidation of these amino acids may contribute to their decline in the IUGR fetus. The rate-limiting step of branched-chain amino acid oxidation is performed by the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKAD), which is regulated by a deactivating kinase. We therefore hypothesized that uteroplacental insufficiency increases BCKAD activity through altered mRNA and protein levels of BCKAD and/or the BCKAD kinase. In IUGR fetal liver, BCKAD activity was increased 3-fold, though no difference in hepatic BCKAD protein or mRNA levels were noted. Hepatic BCKAD kinase mRNA and protein levels were significantly decreased in association with the increase in BCKAD activity. In IUGR fetal skeletal muscle, BCKAD mRNA levels were significantly increased. IUGR skeletal muscle BCKAD protein levels as well as BCKAD kinase mRNA and protein levels were unchanged. We also quantified mRNA levels of two amino acid transporters: LAT1 (system L) and rBAT (cysteine and dibasic amino acids). Both hepatic and muscle LAT1 mRNA levels were significantly increased in the IUGR fetus. We conclude that uteroplacental insufficiency significantly increases hepatic BCKAD activity in association with significantly decreased mRNA and protein levels of the deactivating kinase. We speculate that these changes contribute to the decreased serum levels of branched-chain amino acids seen in the IUGR fetus and may be an adaptation to the deprived milieu associated with uteroplacental insufficiency.

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 (E(1)alpha, E(1)beta and E(2)) in tissues of fed and starved rats; the exceptions were a greater expression of E(1)alpha in the liver and a lower expression of E(1)beta 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 E(1)alpha and E(1)beta. 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.

The key enzyme regulating oxidation of branched-chain keto acids (BCKAs) is BCKA dehydrogenase (BCKAD). We have previously shown that an increase in the activity of this enzyme accounts for the increased oxidation of leucine in the liver of diabetic rats. In the present experiment, we have investigated the mechanisms responsible for this increase in enzyme activity. These studies were performed 96 hours after the withdrawal of insulin therapy in rats made diabetic by an injection of streptozotocin. Diabetes increased the activity state (83% versus 97%, p < .01) as well as the total activity (78 versus 112 nmol/min/mg protein, p < .01) of BCKAD. The increase in the activity state was due to a 60% fall in the BCKAD kinase activity, which was the result of a 50% decrease in its protein mass. A coordinated increase (50%-70%) in protein mass of each BCKAD subunit (E1 alpha, E1 beta, and E2) accounted for the increase in the total activity of BCKAD. We conclude that diabetes increases the hepatic BCKAD activity by increasing its protein mass and also by decreasing that of its associated kinase. These alterations appear to occur posttranscriptionally, since diabetes had no effect on the gene expressions of BCKAD subunits (E1 alpha, E1 beta, and E2) or BCKAD kinase.

Accumulation of products of proteolysis (e.g. dipeptides) in lysosomes may have pathological consequences. In the present experiment we have investigated the existence of a dipeptide transporter in a membrane preparation of liver lysosomes using Gly-3H-Gln as the probe. The results showed that (a) there was transport of Gly-Gln into an osmotically reactive space inside the lysosomal membrane vesicles; (b) transport was stimulated by acidification (pH 5.0) of the external medium; (c) there was a coupling between transport of protons and Gly-Gln with a stoichiometry of 1:1; (d) the presence of both acidic pH and membrane potential was necessary for uphill transport of Gly-Gln; (e) a single transporter with a Km of 4.67 mM mediated the uptake of Gly-Gln; and (f) Gly-Gln uptake was inhibited by dipeptides and tripeptides but not by amino acids. The results suggest the presence of a low affinity proton-coupled oligopeptide transporter in the liver lysosomal membrane which mediates transfer of dipeptides from a region of low dipeptidase activity (intralysosome) to a region of high dipeptidase activity (cytosol). In this manner, the transporter provides an active mechanism for completion of the final stage of protein degradation.

Chronic ethanol intake selectively increases concentrations of branched-chain amino acids (BCAA) in the liver. To determine whether a reduced oxidation plays a role in this effect, we measured substrate flux through branched-chain keto-acid (BCKA) dehydrogenase in livers of rats pair-fed liquid diets containing either 0% or 36% of total calories as ethanol for 21 days. Substrate (1.0 mmol/L ketoisocaproate [KIC]) fluxes in the liver of ethanol-fed and control rats were 225 +/- 18 and 319 +/- 27 mumol/h per whole liver (P < .05), respectively. We then studied whether this effect was due to either ethanol or the products of its metabolism, or to an alteration in the activity of BCKA dehydrogenase. Addition of ethanol (25 to 200 mmol/L) to the perfusion medium had no significant effect on the flux through BCKA dehydrogenase in the liver of control rats. Ethanol-fed rats had lower (P < .01) basal activity (0.84 +/- 0.11 v 1.39 +/- 0.12 U/g liver) and total activity (0.94 +/- 0.11 v 1.42 +/- 0.11 U/g liver) than control rats, but a similar activity state (90% +/- 4% v 99% +/- 4%) of BCKA dehydrogenase. In conclusion, chronic ethanol intake reduces the flux through liver BCKA dehydrogenase by decreasing the basal and total activity of BCKA dehydrogenase and not increasing the conversion of the enzyme to its inactive form.

The present study demonstrates that dexamethasone and 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (CPT-cAMP), a cAMP analog, increase the substrate flux through branched-chain keto acid dehydrogenase (BCKDH) in primary rat hepatocytes cultured in defined medium. Maximum response (2.7-fold increase in flux) was observed when hepatocytes were cultured with 1 microM dexamethasone plus 50 microM CPT-cAMP for 24 h. This increase in the flux rate was accompanied by significant increases in both the basal and total activities of BCKDH (2.2- and 2.0-fold, respectively), without any significant change in the activity state of this enzyme. The increase in BCKDH activity was the result of increased protein mass of E1 alpha (3.2-fold), E1 beta (2.9-fold), and E2 (1.6-fold) subunits of BCKDH, indicating that E2 is the limiting subunit for the expression of BCKDH. The relative abundance of mRNAs encoding the E1 alpha, E1 beta, and E2 subunits of BCKDH increased by 7.4-, 21.7-, and 4.8-fold, respectively. We conclude that increased flux through BCKDH in hepatocytes cultured with dexamethasone and CPT-cAMP is due to increased expression of BCKDH subunit genes. However, nonstoichiometric expression of individual subunits and the corresponding mRNAs suggests regulation of BCKDH also at translational and post-translational steps.

The objective of this study was to compare the metabolic effects of long-term ethanol consumption with oral (Lieber-DeCarli) and enteral feeding techniques. Enteral feeding allowed administration of greater amounts of energy and nutrients. After 21 days of treatment using the Lieber-DeCarli technique, the ethanol-treated rats had the following significant (P less than 0.05) differences from pair-fed controls: lower cumulative nitrogen balance (days 5-21; 2.8 +/- 0.1 g N vs. 3.5 +/- 0.1 g N), lower protein content of gastrocnemius muscle (289 +/- 17 mg vs. 358 +/- 11 mg) and intestinal mucosa (461 +/- 19 mg vs. 577 +/- 40 mg), higher plasma leucine concentration (147 +/- 8 mumol/L vs. 102 +/- 8 mumol/L), higher liver protein content (2222 +/- 122 mg vs. 1679 +/- 58 mg), and higher liver triglyceride concentration (38.4 +/- 2.8 mg/g vs. 8.7 +/- 1.0 mg/g). When rats received the same amount of nitrogen (1.5 g.kg-1.day-1) and ethanol (13 g.kg-1.day-1) but 16.3% more energy and nutrients by a surgically implanted gastric cannula (enterally fed), the effects of ethanol on nitrogen balance, tissue protein content, plasma leucine concentration, and liver triglyceride concentration were similar to those observed in the rats fed orally. It is concluded that the metabolic effects observed using the Lieber-DeCarli feeding technique are due to ethanol per se and not the synergism of ethanol and undernutrition as recently suggested.

1. Rat liver was fractionated into peroxisomes and mitochondria and branched-chain keto acid (BCKA) dehydrogenase activity was measured. 2. All BCKA dehydrogenase activity was associated with the mitochondrial fraction and none with the peroxisomal fraction. 3. BCKA dehydrogenase was also not detected in hepatic peroxisomes of rats treated with clofibrate which induces several peroxisomal enzymes. 4. Hepatic peroxisomes from rabbit, hamster and dog also did not show any BCKA dehydrogenase activity. 5. We conclude that mammalian hepatic peroxisomes do not contain BCKA dehydrogenase.

We have investigated whether hepatic peroxisomes are capable of synthesizing carnitine. When purified peroxisomes were incubated with gamma-butyrobetaine, a precursor of carnitine, formation of carnitine was observed. These results indicate that peroxisomes contain gamma-butyrobetaine hydroxylase, the enzyme which catalyzes the final step in the biosynthesis of carnitine. This enzyme was previously believed to be present only in the cytosol. gamma-Butyrobetaine hydroxylase activity in peroxisomes was not due to cytosolic contamination as evaluated by marker enzyme analysis. When proliferation of peroxisomes was induced by clofibrate treatment, gamma-butyrobetaine hydroxylase/mass liver increased by 7.6-fold and the specific activity by 2.5-fold. We conclude that hepatic peroxisomes synthesize carnitine and this synthesis becomes substantial under conditions of peroxisomal proliferation.

The specific activity of leucine transaminase was measured in supernatants of liver, skeletal muscle (gastrocnemius), and kidney homogenates obtained from fed, starved, and protein-deprived rats. After 12 h of starvation, there were slight reductions in leucine transaminase activity of both muscle and kidney tissues. When starvation was prolonged to 1 full day, the activity of this enzyme increased by approximately twofold in both muscle and kidney. Prolongation of fasting to 5 days resulted in an additional increase in specific activity of leucine transaminase in muscle. During the entire 5 days of starvation, leucine transaminase activity remained unaltered in liver of starved rats. Protein deprivation for 1 or 5 days resulted in significant reductions in specific activity of leucine transaminase in skeletal muscle. Protein deprivation did not produce a remarkable effect on the activity of this enzyme in kidney or liver tissue. The results of this study, together with those previously obtained, indicate that within our experimental conditions increased oxidation of leucine in skeletal muscle of starved rats is not initially related to an alteration in activity of leucine transaminase. When caloric deficiency is prolonged, the potential for transamination is also increased. These adaptive changes increase the ability of skeletal muscle to use leucine as a metabolic fuel.

To investigate dipeptide assimilation by the liver, a series of interrelated experiments were performed in rats. Partial hepatectomy prolonged the plasma half-life (min) of Gly-Ala (3.42 +/- 0.22 versus 4.90 +/- 0.35, p less than 0.05) but had no significant effect on plasma half-life of Gly-Leu, Gly-Pro, or Gly-Sar. We then investigated the rate of disappearance (mumol X (g liver X h)-1) of the above four dipeptides (initial concentration = 1 mM) from the medium during isolated liver perfusion. The order of dipeptide disappearance was: Gly-Leu (8.75 +/- 0.65) greater than Gly-Ala (3.36 +/- 0.46) greater than Gly-Pro (1.29 +/- 0.54) greater than Gly-Sar (0.35 +/- 0.12). This order of dipeptide disappearance corresponded exactly to the order of the rates of glycine accumulation in the medium during liver perfusion with the four dipeptides. Addition of glucagon had no effect on the disappearance rate of Gly-Ala from the medium, but reduced accumulation rates of glycine (3.39 +/- 0.30 versus 1.42 +/- 30, p less than 0.01) and alanine (4.42 +/- 0.66 versus 1.35 +/- 0.39, p less than 0.01). Finally, we found that hydrolysis by the liver plasma membranes and/or perfusion medium accounted for disappearance of dipeptides. In conclusion, the liver does not appear to have a transport system for dipeptides, but assimilates dipeptides by extracellular hydrolysis. Hydrolysis is achieved by enzymes either located on the plasma membranes or released from the cytosol. The amino acid residues released as the result of dipeptide hydrolysis are then taken up by the liver.

Peptide hydrolase activities against glycyl-L-leucine and glycylglycine were investigated in the soluble fractions of blood, liver, kidney cortex, skeletal muscle (gastrocnemius), and jejunal and ileal mucosa of rats. The maximal hydrolase activity in each tissue was determined when the incubation conditions, such as time, pH, substrate and enzyme concentrations, and ionic requirements were optimal. The kinetic constants (apparent Km and Vmax) were determined from Lineweaver-Burk double reciprocal graphs. Maximal hydrolysis rates against both dipeptides were many times greater by kidney and intestinal segments than by those of muscle, liver, or blood. The order of Vmax for hydrolase activity against glycylleucine was kidney greater than ileum greater than jejunum greater than liver greater than muscle greater than blood, and the order against glycylglycine was ileum greater than kidney greater than jejunum greater than liver greater than muscle. Those tissues that had the lowest Vmax values (liver and muscle), when calculated on the basis of tissue weights, were shown to have activities comparable to the others. Results of this study are consistent with our previous findings that: 1) glycylleucine half-life in plasma is shorter than that of glycylglycine, and 2) disappearance rates of these dipeptides from plasma are not affected by nephrectomy or enterectomy.

Chronic clofibrate intake, on occasion, results in a muscular syndrome in man. We have investigated the effects of chronic clofibrate administration in rats on the electrical activity of a skeletal muscle (gastrocnemius), its composition, and its oxidation of palmitate and glucose. These effects have been compared with those in the liver. Clofibrate administration altered electromyographic pattern of gastrocnemius muscle (characteristic of myotonia), decreased its protein content, and impaired its oxidation of palmitate and glucose. These effects were quite different in the liver, because clofibrate intake increased the liver protein content and oxidation of palmitate without affecting the oxidation of glucose by this tissue. Whereas chronic clofibrate administration markedly increased the concentration of carnitine as well as the activity of mitochondrial carnitine palmitoyl-transferase in the liver, it decreased the activity of this enzyme in the gastrocnemius muscle without a significant effect on carnitine concentration in this tissue. Greater in vivo fatty acid oxidation by clofibratefed than by control rats was evidenced (a) by greater rate of production of (14)CO(2) in the expired air after injection of a tracer dose of [(14)C]palmitate and (b) by greater plasma and tissue concentrations of ketone bodies. We conclude that (a) paradoxical effects of clofibrate on fatty acid oxidation by the liver and skeletal muscle are related to changes in the activity of carnitine acyltransferase, (b) an increase in hepatic fatty acid oxidation may contribute to hypolipidemic effect of clofibrate, and (c) impairment of fatty acid and glucose oxidation by the muscle may be a factor in the development of muscular syndrome in patients receiving clofibrate treatment.

The authors' previous studies have shown that hepatic steatosis of chronic ethanol ingestion in rats can be prevented by adding pyruvate, dihydroxyacetone, and riboflavin to their diet. In this study, the authors investigated the effect of chronic ethanol ingestion, with or without addition of the above metabolites to the diet, on protein and amino acid concentrations in tissues. Rats (120 g) were divided into three groups and fed isocalorically one of the fellowing diets for 30 days: control diet (28% fat, 15% protein, and 57% carbohydrate), ethanol diet (28% fat, 15% protein, 23% carbohydrate, and 24% ethanol), and metabolite diet (ethanol diet plus pyruvate, dihydroxyacetone, and riboflavin). Chronic ethanol ingestion reduced growth of muscle and intestinal mucosa without affecting that of liver and kidney. Among the 15 amino acids measured, chronic ethanol ingestion had the most consistent effect on plasma and tissue concentrations of leucine, alanine and alpha-amino-n-butyrate. The concentration of leucine was increased in muscle, liver, and plasma; that of alpha-amino-n-butyrate was increased in muscle and plasma, whereas that of alanine was decreased in plasma and liver. Addition of pyruvate, dihydroxyacetone, and riboflavin to the ethanol diet either totally or partially prevented ethanol-induced changes in plasma and tissue concentrations of amino acids despite similarity in plasma ethanol levels. Although these metabolites prevented the inhibition of the growth of intestinal mucosa, they were ineffective in blunting the effect of ehtanol on the skeletal muscle. This latter observation suggests that the mechanism of ethanol-induced inhibition of tissue growth is not the same for these tissues.

We have investigated the influence of amino acid residues on hepatic clearance of oligopeptides by determining the rate of disappearance (nmol.(min.g liver)-1) of selective oligopeptides from the medium during isolated rat liver perfusion. (a) N terminus: the rate of disappearance of Ala-Leu was greater (p less than 0.01) than those of Gly-Leu, Phe-Leu, and Arg-Leu (208 +/- 13, 135 +/- 13, 116 +/- 12, and 127 +/- 12, respectively). (b) C terminus: the rate of disappearance of Leu-Ala (244 +/- 18) was significantly greater (p less than 0.01) than that of Leu-Gly (145 +/- 16). (c) Number of residues: with each increase in the number of alanine residues (2-4) there was a significant increase in the rate of peptide disappearance, and conversely, with each increase in the number of glycine residues (2-6) there was a significant decrease in the rate of peptide disappearance. Further studies showed no peptide transport by isolated liver plasma membrane vesicles and no significant correlation between the rates of peptide disappearance and hydrolase activities of the perfusion medium but highly significant correlation with hydrolase activity of plasma membrane. We conclude that certain amino acid residues, such as alanine, enhance hepatic clearance of oligopeptides by increasing their affinity as substrates for plasma membrane peptide hydrolases.

Oligopeptides may enter the bloodstream from endogenous and exogenous sources. Using an organ-balance technique in conscious dogs, we investigated the role of individual organs in removal of two model oligopeptides (glycylleucine and glycylglycine) from plasma under steady-state conditions. Despite an identical infusion rate, arterial concentration of glycylglycine was twofold greater than that of glycylleucine. This appeared to be a result of greater fractional extraction of glycylleucine than glycylglycine by organs. Although all of the organs examined participated in removal of dipeptides from plasma, their roles varied. Liver, kidney, muscle, and gut accounted for the disappearance of 25, 24, 12, and 10% of the infused amount of glycylleucine, respectively. With glycylglycine as the substrate, disappearance across kidney accounted for 37% of the infused amount, whereas muscle, liver, and gut accounted for 18, 15, and 11%, respectively. Finally, we investigated glycine and leucine balances across organs with infusion of these amino acids in free and dipeptide forms. Glycine and leucine balances were uniquely more positive across muscle during the infusion of glycylleucine than the corresponding amino acid mixture. The possible mechanisms included release of products of glycylleucine hydrolysis by all organs except muscle. We conclude that molecular structure influences the organ extraction of dipeptides; if extraction, particularly by the liver, is not sufficiently rapid, kidney assumes a greater role than other organs in dipeptide removal from plasma.

The role of liver, muscle, kidney and gut in the assimilation of intravenously administered glycylleucine was investigated in 8 mongrel dogs. The rates of disappearance of glycylleucine during its passage across liver, muscle, kidney and gut were 1487 +/- 80, 740 +/- 216, 1436 +/- 115, and 602 +/- 103 mumol/(min x kg B.W.), respectively. The infusion of glycylleucine greatly altered the fluxes of glycine and leucine across these organs. The major alterations included increases in the uptake of glycine by the liver and that of leucine by the muscle, and increases in release of leucine by liver and kidney. We conclude that all organs are involved in the assimilation of intravenously administered glycylleucine, but with varying importance. Although liver and kidney appear to be the dominant organs for the assimilation of glycylleucine, the metabolism of glycine is chiefly accomplished in the liver, and that of leucine is chiefly accomplished in the muscle.

The effect of L-carnitine (0.5-2.0 mM) on the rates of alpha-decarboxylation of 1-14C-labeled branched-chain amino acids by gastrocnemius muscle and liver homogenates of fed rats was investigated. Carnitine increased the rate of alpha-decarboxylation of leucine (125%) and valine (28%) by muscle, but it was without effect on the oxidation of these amino acids by liver. Carnitine increased the rate of alpha-decarboxylation of alpha-ketoisocaproate by both tissues. This effect was more pronounced in muscle (130% increase) than in liver (41% increase). The activity of carnitine acyltransferase, with isovaleryl-CoA as a substrate, was 18 times higher in muscle mitochondria than in liver mitochondria. Both starvation and diabetes increased the rate of alpha-decarboxylation of leucine by muscle without having a remarkable effect on the concentration of carnitine or the activity of carnitine acyltransferase. We conclude that: a) carnitine stimulates decarboxylation of branched-chain amino acids by increasing the conversion of their ketoanalogues into carnitine esters, b) a greater carnitine acyltransferase activity in muscle than in liver may be responsible for the greater carnitine effect in muscle, c) carnitine does not appear responsible for the enhancement of leucine oxidation by muscle of starved and diabetic rats.

Our previous studies have shown that treatment of rats with clofibrate, a hypolipidemic drug, greatly increases the total concentration of carnitine in the liver (H. S. Paul and S. A. Adibi, J. Clin. Invest. 64: 405-412, 1979). In the present experiment we have investigated some possible mechanisms to account for this increase. Clofibrate treatment (30 mg/100 g rat/day for 2 wk) increased significantly the concentration (nmol/g, mean +/- SE, 6 rats) of both free (289 +/- 21 vs. 1,747 +/- 131) and acylcarnitine (87 +/- 11 vs. 412 +/- 42). These increases were not the result of redistribution of carnitine among tissues or due to a decrease in urinary excretion. In view of previous observations that thyroid hormones increase the hepatic concentrations of carnitine, and clofibrate treatment causes a hyperthyroid state in the liver, we investigated the effect of clofibrate in thyroidectomized rats. Clofibrate treatment of thyroidectomized rats also increased the concentration of free (423 +/- 25 vs. 1,460 +/- 123) and acylcarnitine (35 +/- 6 vs. 305 +/- 31) in the liver. Finally, clofibrate treatment significantly increased the urinary excretion of trimethyllysine, a precursor of carnitine (31 +/- 3 vs. 47 +/- 4 nmol/mg creatinine, mean +/- SE, 5 rats). Our data suggest that clofibrate treatment stimulates hepatic synthesis of carnitine by increasing the availability of its precursor, trimethyllysine. This effect of clofibrate is independent of thyroid hormone.