Uteroplacental insufficiency alters liver and skeletal muscle branched-chain amino acid metabolism in intrauterine growth-restricted fetal rats.

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.

Protein expressions of branched-chain keto acid dehydrogenase subunits are selectively and posttranscriptionally altered in liver and skeletal muscle of starved rats.

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.

Characterization of an oligopeptide transporter in renal lysosomes.

Renal lysosomes play a major role in catabolism of plasma proteins. Final products of this catabolism include dipeptides and tripeptides that must be exported to the cytosol for hydrolysis. The aim of the present study was to determine whether an oligopeptide transporter is present in the renal lysosomal membrane that could mediate this export. The existence of an oligopeptide transporter was probed with the uptake of glycylglutamine (Gly-Gln) by membrane vesicles prepared from renal lysosomes. Kinetic analysis showed the presence of a single transporter with a K(m) of 8.77 mM for the uptake of Gly-Gln. The Gly-Gln uptake was energized by the imposition of an inwardly directed proton gradient (pH(out) 5.0/pH(in) 7.3) and membrane potential (outside positive/inside negative) resulting in overshoot. The Gly-Gln uptake was inhibited by the presence of dipeptides and tripeptides, but not amino acids. Western blot analysis of lysosomal membrane proteins with Pept-1 (an oligopeptide transporter) antibody as the probe showed the presence of an immunoreactive protein. This immunoreaction was abolished when the antiserum was preabsorbed with the Pept-1 epitope (0.5 microg/ml). In conclusion, the present data show the existence of a low-affinity dipeptide transporter in the renal lysosomal membrane that appears to belong to the Pept family of transporters. The function of this transporter appears to be to prevent accumulation of dipeptides in renal lysosomes.

Inverse alterations of BCKA dehydrogenase activity in cardiac and skeletal muscles of diabetic rats.

Rat cardiac and skeletal muscles, which have been used as model tissues for studies of regulation of branched-chain alpha-keto acid (BCKA) oxidation, vary greatly in the activity state of their BCKA dehydrogenase. In the present experiment, we have investigated whether they also vary in response of their BCKA dehydrogenase to a metabolic alteration such as diabetes and, if so, to investigate the mechanism that underlies the difference. Diabetes was produced by depriving streptozotocin-treated rats of insulin administration for 96 h. The investigation of BCKA dehydrogenase in the skeletal muscle (gastrocnemius) showed that diabetes 1) increased its activity, 2) increased the protein and gene expressions of all of its subunits (E(1)alpha, E(1)beta, E(2)), 3) increased its activity state, 4) decreased the rate of its inactivation, and 5) decreased the protein expression of its associated kinase (BCKAD kinase) without affecting its gene expression. In sharp contrast, the investigation of BCKA dehydrogenase in the cardiac muscle showed that diabetes 1) decreased its activity, 2) had no effect on either protein or gene expression of any of its subunits, 3) decreased its activity state, 4) increased its rate of inactivation, and 5) increased both the protein and gene expressions of its associated kinase. In conclusion, our data suggest that, in diabetes, the protein expression of BCKAD kinase is downregulated posttranscriptionally in the skeletal muscle, whereas it is upregulated pretranslationally in the cardiac muscle, causing inverse alterations of BCKA dehydrogenase activity in these muscles.

Functional and molecular expression of intestinal oligopeptide transporter (Pept-1) after a brief fast.

The intestinal oligopeptide transporter, cloned as Pept-1, has major roles in the assimilation of dietary proteins and absorption of peptidomimetic medications. The initial aim of the present experiment was to investigate whether the functional expression of this transporter is affected by dietary intake. Functional expression was determined as the rate of uptake of glycylglutamine (Gly-Gln) by brush-border membrane vesicles (BBMVs) prepared from the jejunum of fed and fasted rats. Surprisingly, the rate of dipeptide uptake was greatly increased after 1 day of fasting. The subsequent aim of the experiment became the investigation of the mechanism of this alteration in transport, which showed that 1 day of fasting increased (1) the maximal Gly-Gln uptake (Vmax) by twofold without changing the Km of Gly-Gln uptake by BBMVs, (2) the amount of intestinal oligopeptide transporter (Pept-1) protein by threefold in the brush-border membrane, and (3) the abundance of Pept-1 mRNA by threefold in the intestinal mucosa. We conclude that 1 day of fasting increases dipeptide transport in rat intestine by increasing the population of Pept-1 in the brush-border membrane. The mechanism appears to be an increase in Pept-1 gene expression.

Hormonal regulation of oligopeptide transporter pept-1 in a human intestinal cell line.

The intestinal oligopeptide transporter (cloned as Pept-1) has major roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. In this experiment, we investigated whether insulin has such a role. We used a human intestinal cell monolayer (Caco-2) as the in vitro model of human small intestine and glycylglutamine (Gly-Gln) as the model substrate for Pept-1. Results showed that addition of insulin at a physiological concentration (5 nM) to incubation medium greatly stimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blocked when genistein, an inhibitor of tyrosine kinase, was added to incubation medium. Studies of the mechanism of insulin stimulation showed the following. 1) Stimulation occurred promptly (30-60 min) after exposure to insulin. 2) There was no significant change in the Michaelis-Menten constant of Gly-Gln transport, but there was a nearly twofold increase in its maximal velocity. 3) Insulin effect persisted even when Golgi apparatus, which is involved in trafficking of newly synthesized Pept-1, was dismantled. 4) However, there was complete elimination of insulin effect by disruption of microtubules involved in trafficking of preformed Pept-1. 5) Finally, with insulin treatment, there was no change in Pept-1 gene expression, but the amount of Pept-1 protein in the apical membrane was increased. In conclusion, the results show that insulin, when it binds to its receptor, stimulates Gly-Gln uptake by Caco-2 cells by increasing the membrane population of Pept-1. The mechanism appears to be increased translocation of this transporter from a preformed cytoplasmic pool.

Mechanism of dipeptide stimulation of its own transport in a human intestinal cell line.

The initial objective of this study was to investigate whether the presence of dipeptide in the culture medium stimulates the uptake of dipeptide by a human intestinal cell line that expresses the oligopeptide transporter (Pept-1). The results showed that addition of glycylsarcosine (Gly-Sar) for 24 hr to the culture medium significantly increased the rate of glycylglutamine (Gly-Gln) uptake by Caco-2 cells. Furthermore, this stimulation in transport was also observed when Cefadroxil (beta-lactam antibiotic) instead of Gly-Gln was used as a probe but did not occur when Gly-Sar was added to the culture medium for only 2 hr or when Gly-Sar was substituted by a corresponding mixture of glycine plus sarcosine. The subsequent objective of the study was to investigate the mechanism of stimulation in transport described earlier. The results showed that the addition of Gly-Sar for 24 hr to the culture medium: (1) increased the Vmax of Gly-Gln transport by two-fold without affecting its Km, (2) increased the protein mass of Pept-1 by more than two-fold, (3) increased the abundance of Pept-1 mRNA by three-fold, and (4) had no effect on Gly-Gln transport when an inhibitor of trans-Golgi network (brefeldin) was added to the culture medium, but still increased the abundance of Pept-1 mRNA. In conclusion, the results show that dipeptides stimulate their own transport by increasing the membrane population of Pept-1. The molecular mechanism appears to be an increase in expression of the gene encoding Pept-1. A therapeutic application of the present results is that if bioavailability of orally administered peptidomimetic drugs is limited, patients may be tried on a high-protein diet to enhance their absorption.

Posttranscriptional alterations in protein masses of hepatic branched-chain keto acid dehydrogenase and its associated kinase in diabetes.

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.

The oligopeptide transporter (Pept-1) in human intestine: biology and function.

The oligopeptide transporter (Pept-1), which is located in the intestinal brush border membrane, provides a major mechanism for protein absorption in the human intestine. Expression cloning of the gene encoding Pept-1 has predicted a 78,810-kilodalton protein consisting of 708 amino acid residues and possessing 12 putative membrane-spanning domains. The characterization of its function by in vivo and in vitro studies has shown that (1) it transports dipeptides and tripeptides but not free amino acids or peptides with more than three amino acid residues, and (2) its driving force for uphill transport requires proton binding and presence of an inside-negative membrane potential. There has also been cloning of a membrane protein (HPT-1) which appears to be associated with the oligopeptide transporter. However, the nature of association has not yet been determined. A human intestinal cell line (Caco-2), which expresses Pept-1, has been used to investigate the effects of metabolic and pathological factors on dipeptide transport. These studies suggest that the insulin stimulates dipeptide transport by increasing membrane insertion of oligopeptide transporter from a preformed cytoplasmic pool, and cholera toxin decreases dipeptide transport by inhibiting the activity of Pept-1 through an increase in the intracellular concentration of adenosine 3',5'-cyclic monophosphate. Lastly, Pept-1 seems to play important roles in nutritional and pharmacological therapies; for example, it has allowed the use of oligopeptides as a source of nitrogen for enteral feeding and the use of oral route for delivery of peptidomimetic drugs such as beta-lactam antibiotics.

Renal assimilation of oligopeptides: physiological mechanisms and metabolic importance.

Assimilation of systemic oligopeptides (di- and tripeptides) is largely a function of kidneys. The most specific and unique mechanism utilized for the performance of this renal function is transport, followed by intracellular hydrolysis and then release of constituent amino acids to the systemic circulation. Among tissues examined (liver, kidney, intestine, and muscle), kidney is the only tissue capable of accumulating dipeptides in concentrations that are greater than their plasma concentrations. Kidney also is the tissue with the highest cytoplasmic dipeptidase activity. Intracellular accumulation is mediated by two transporters (Pept-1 and Pept-2), both of which have been recently cloned. These transporters use dipeptides and tripeptides as substrates and rely on protons and membrane potential for their driving force. Pept-1 is a low-affinity, high-capacity transporter, and Pept-2 is a high-affinity, low-capacity transporter. The nutritional and metabolic regulation of renal assimilation of oligopeptides is suggested by the selective decrease in dipeptide balance across the kidneys of starved human subjects and by the insulin stimulation of dipeptide transport by a renal cell line. Peptiduria has been observed in a variety of diseases, but the mechanism, except in genetic diseases affecting hydrolysis of oligopeptides, is not known. Finally, the capacity for active transport of oligopeptides and peptidomimetic drugs enables kidneys to play major roles in nutritional and pharmacological therapies.

An active mechanism for completion of the final stage of protein degradation in the liver, lysosomal transport of dipeptides.

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.

Mechanism of clearance and transfer of dipeptides by perfused human placenta.

Glycylglutamine (Gly-Gln) is stable source of glutamine for parenteral nutrition. In the present study we have investigated whether this dipeptide is transferred intact across the human placenta. Although after 90 min of placental perfusion there was almost complete disappearance of Gly-Gln (100 microM) from the maternal compartment, only a small concentration of this dipeptide (< 6 microM) appeared in the fetal compartment. To investigate whether this transfer was due to transcellular transport, brush-border membrane vesicles of the human placenta were probed with [3H]Gly-Gln, which showed no uptake. To investigate whether hydrolysis was the mechanism of disappearance of Gly-Gln, the perfusion study was repeated with glycylsarcosine (Gly-Sar), which is resistant to hydrolysis. In sharp contrast to Gly-Gln, after 90 min of perfusion nearly 80% of Gly-Sar remained in the perfusate (half-life of 24 vs. 235 min). The rest of the Gly-Sar was recovered intact in the fetal compartment. The addition of Gly-Gln to the maternal compartment increased the accumulation of glycine, but not glutamine, in both the maternal and fetal compartments. In conclusion, our data suggest that 1) the mechanism of clearance of Gly-Gln by perfused human placenta is largely hydrolysis, whereas that of Gly-Sar is largely passive diffusion, and 2) the placenta has a greater preference for glutamine than for glycine.

Alteration in gene expression of branched-chain keto acid dehydrogenase kinase but not in gene expression of its substrate in the liver of clofibrate-treated rats.

We previously showed that the oxidation of branched-chain amino acids is increased in rats treated with clofibrate [Paul and Adibi (1980) J. Clin. Invest. 65, 1285-1293]. Two subsequent studies have reported contradictory results regarding the effect of clofibrate treatment on gene expression of branched-chain keto acid dehydrogenase (BCKDH) in rat liver. Furthermore, there has been no previous study of the effect of clofibrate treatment on gene expression of BCKDH kinase, which regulates the activity of BCKDH by phosphorylation. The purpose of the present study was to investigate the above issues. Clofibrate treatment for 2 weeks resulted in (a) a 3-fold increase in the flux through BCKDH in mitochondria isolated from rat liver, and (b) a modest but significant increase in the activity of BCKDH. However, clofibrate treatment had no significant effect on the mass of E1 alpha, E1 beta, and E2 subunits of BCKDH or the abundance of mRNAs encoding these subunits. On the other hand, clofibrate treatment significantly reduced the activity, the protein mass and the mRNA levels of BCKDH kinase in the liver. In contrast to the results obtained in liver, clofibrate treatment had no significant effect on any of these parameters of BCKDH kinase in the skeletal muscle. In conclusion, our results show that clofibrate treatment increases the activity of BCKDH in the liver and the mechanism of this effect is the inhibition of gene expression of the BCKDH kinase.

Chronic ethanol intake reduces the flux through liver branched-chain keto-acid dehydrogenase.

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.

Selective effect of zinc on uphill transport of oligopeptides into kidney brush border membrane vesicles.

Based on the involvement of zinc in hydrolysis of peptides, we hypothesized that Zn2+ may also play a role in peptide transport. To investigate this hypothesis, kidney brush border membrane vesicles (BBMV) were incubated for 30 min with different concentrations of ZnSO4 before use in uptake studies. This incubation increased by twofold the overshoot uptake of 3H-Gly-L-Gln, D-Leu-125I-Tyr and 3H-cephalexin (all high-affinity substrates for the oligopeptide/H+ symporter) without affecting passive and/or facilitated diffusion of these substrates. Zinc had no effect on the uptake of either glutamine or glucose by kidney BBMV. Among a group of metal ions (cobalt, iron, copper, cadmium, and manganese), only manganese and copper substantially stimulated the activity of the oligopeptide/H+ symporter. DTPA (a complexing agent) inhibited dipeptide uptake, which was reversed by the addition of zinc to the BBMV. Zinc treatment of BBMV reduced the EC50 value of inhibition of 3H-Gly-L-Gln uptake by unlabeled Gly-L-Gln by twofold (90 +/- 8 vs. 45 +/- 4 microM). Similarly, zinc treatment of BBMV reduced the EC50 value for inhibition of D-Leu-125I-Tyr uptake by bestatin from 80 +/- 4 to 40 +/- 3 mM. In conclusion, the data show that zinc has a selective effect on transport of nutrients into kidney BBMV. It stimulates uphill transport of oligopeptides by a modification of their affinity for the binding site of the membrane transporter.

Regulation of gene expression of branched-chain keto acid dehydrogenase complex in primary cultured hepatocytes by dexamethasone and a cAMP analog.

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.

Functional separation of dipeptide transport and hydrolysis in kidney brush border membrane vesicles.

Dipeptides serve as substrates for both transport and hydrolytic processes. This has caused uncertainty as to whether different systems mediate both processes, and if so, whether they can be functionally separated. To investigate these problems, we determined the effects of a series of compounds previously characterized as aminopeptidase (AP) inhibitors on transport and hydrolytic activities of the kidney brush border membrane. The substrate used for assaying transport activity was Gly-Gln whereas Leu-, Arg-, and Glu-nitroanilides, as well as Gly-Tyr and Ala-Tyr, were used in assaying hydrolytic activity. The AP inhibitors, arphamenines A and B and bestatin, strongly inhibited transport by the oligopeptide/H+ symporter (EC50 values of 15 to 67 microM). The mechanism of inhibition appeared to be competition for the binding site of the symporter. In contrast, leucinethiol, leuhistin, and amastatin had little or no effect on dipeptide transport (EC50 values of 4 to more than 50 mM). Arphamenine and leucinethiol, in concentrations as high as 100 microM, were found to be either ineffective or weak inhibitors of membrane-associated hydrolysis. In contrast, amastatin and leuhistin, in concentrations as low as 20 microM, almost completely inhibited dipeptide hydrolysis. These results show that dipeptide hydrolysis can be selectively suppressed by either amastatin or leuhistin and dipeptide transport by arphamenine. Furthermore, the results provide new insight into the structural features of substrates that are recognized at the binding site of the oligopeptide/H+ symporter.

Transport of beta-lactam antibiotics in kidney brush border membrane. Determinants of their affinity for the oligopeptide/H+ symporter.

This study was designed to determine whether beta-lactam antibiotics (cephalosporins and penicillins) are all substrates for the renal oligopeptide/H+ symporter and, if so, whether the transport system discriminates among the numerous beta-lactam antibiotics. We used [3H]glycylglutamine, [3H]cephalexin, and [3H]-ampicillin as probes for the transport of oligopeptides, cephalosporins, and penicillins in kidney brush border membrane vesicles, respectively. Among the beta-lactam antibiotics, only those with an alpha-amino group in the phenylacetamido moiety were found to interact with the oligopeptide/H+ symporter. Aminocephalosporins displayed high affinities (KiS generally < 250 microM), whereas aminopenicillins displayed low affinities (Ki 0.78-3.03 mM). These differences in affinities appeared to be a consequence of conformational features of the substrates, especially the sterical location of the carboxy group. The affinities of aminolactams for the oligopeptide/H+ symporter were, furthermore, related to the hydrophobicity of the phenylglycyl chains and the substituents attached to the thiazolidine and dihydrothiazine ring. In sharp contrast to the uptake of [3H]glycylglutamine and [3H]cephalexin, the uptake of [3H]ampicillin was not dependent on a pH gradient and was inhibited by various beta-lactam antibiotics, whether or not they contained an alpha-amino group. Our data suggest that: (a) the transport of aminocephalosporins is largely mediated by the oligopeptide/H+ symporter, which is highly influenced by the substrate structure; and (b) penicillins are transported by another system, which is less discriminative with respect to substrate structure.

Leucine metabolism during chronic ethanol consumption.

Chronic ethanol consumption is known to increase plasma concentrations of branched-chain amino acids (BCAA) in rats and man, but the mechanisms of this effect are not known. Chronic ethanol consumption may increase levels of BCAA by altering protein turnover and/or by affecting the oxidation of BCAA. These possibilities were investigated in rats pair-fed liquid diets containing either 0% or 36% of total calories as ethanol for 21 days. In the fed state, ethanol-treated rats had a plasma ethanol level of 20 +/- 5 mmol/L and twofold increases in BCAA concentrations in plasma. There were also significant increases (37% to 63%) in muscle, liver, and jejunal mucosa BCAA concentrations. Chronic ethanol consumption significantly increased whole-body rates (mumol/100 g/h) of leucine turnover (73.8 +/- 7.5 v 104 +/- 5.6, P < .01) and oxidation (12.0 +/- 1.7 v 17.7 +/- 1.1, P < .05). In contrast, it significantly decreased leucine incorporation (nmol/mg protein/240 min) into both muscle (0.61 +/- 0.07 v 0.35 +/- 0.05, P < .01) and liver (13.25 +/- 1.40 v 6.78 +/- 0.98, P < .01) proteins. Incorporation of leucine into the mucosal proteins of jejunum (17.42 +/- 1.42 v 15.85 +/- 1.90, P = NS) was not significantly altered by ethanol. These results suggest that reduced protein synthesis and/or increased protein breakdown may account for the elevated tissue BCAA concentrations in chronic ethanol consumption. The consequences of these increased tissue concentrations are increases in tissue oxidation and plasma concentrations of BCAA.

Dipeptides in parenteral nutrition: from basic science to clinical applications.

The use of intravenous dipeptides shows great promise as an avenue for the provision of amino acids that may otherwise be difficult to deliver via nutrient infusions. The physical/chemical properties and metabolism of numerous dipeptides have now been explored in experimental and human studies. It has been found that these agents have the capacity to spare nitrogen and support serum protein levels in a fashion equivalent to that of intravenous free amino acids. An additional benefit is the ability to deliver certain amino acids that are relatively unstable or poorly soluble in aqueous solutions. These various aspects of intravenous dipeptides are considered in this review.