Siamak A. Adibi

Metabolism of branched-chain amino acids in altered nutrition

Plasma concentrations of the branched-chain amino acids (leucine, isoleucine, and valine) are more prominently affected than the concentrations of other amino acids by changes in dietary-caloric, protein, fat, and carbohydrate-intake in man. For example, within a day of starvation or protein deprivation, there are increases or decreases, respectively, in concentrations of these amino acids in the plasma of healthy human volunteers. The cellular mechanisms of these changes have been investigated in rats, since the changes in the plasma branched-chain amino acid concentrations in response to the previously stated dietary alterations are similar to those found in man. Among the tissues studied (liver, skeletal muscle, heart, kidney, and intestine) only liver and the skeletal muscle exhibit changes in branched-chain amino acid concentrations in response to dietary alteation. Changes in plasma concentrations appear to reflect more intimately those of the muscle than theliver. After 8 days of starvation, there is a 25% decrease in the muscle protein, but after 8 days of protein deprivation, there is no significant change in the muscle mass. Increases in concentrations of branched-chain amino acids in the muscle are much smaller than the amounts of these amino acids lost as protein constituents form the muscle during fasting. Changes in tissue transport, transamination, oxidation, or metabolic conversions of branched-chain amino acids in tissues. It is concluded that increased muscle protein breakdown, which provides substrates for enhanced gluconeogenesis in the liver and enhanced branched-chain amino acid oxidation in the muscle, is the major mechanism of hyperbranched-chain aminoacdemia in starvation. On the other hand, the principal factors in the development of hypobranched-chain aminoacidemia during protein deprivation are absence of exogenous amino acids as well as curtailed muscle protein breakdown.

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.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.

Development of hepatic cholestasis and fibrosis in patients with massive loss of intestine supported by prolonged parenteral nutrition.

We studied the effect of 1 yr of parenteral nutrition on liver function tests and, when indicated, liver histology and ultrastructure of 18 patients with no (n = 6), modest (n = 6), and massive (n = 6) loss of intestine. The resection was for Crohns disease and infarction, respectively. The liver function tests remained normal in all patients with no loss and modest loss of intestine. Four patients with massive loss of intestine, 4-10 mo after initiation of parenteral nutrition, began to develop progressive, marked increases in serum alkaline phosphatase (2-10 times normal), glutamic oxaloacetic transaminase (7-20 times normal), and glutamic pyruvic transaminase (5-14 times normal) activity levels, and bilirubin concentration (5-22 times normal). Light microscopic examination of liver showed cholestasis, bile ductular proliferation, periportal inflammation, fibrosis, and mild steatosis. Electron microscopic examination of liver showed cholestasis with nonspecific organelle changes. None of the patients had any evidence of extrahepatic obstruction. Our data suggest that massive loss of intestine is a contributing factor to hepatic cholestasis and fibrosis in patients maintained on prolonged parenteral nutrition.

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.

Characteristics and mechanism of glutamine-dipeptide absorption in human intestine

Using in vivo and in vitro techniques, the mechanism by which intestinal mucosa obtains glutamine from luminal oligopeptides was investigated in humans. The rate of hydrolysis by mucosal brush border membrane was more than threefold greater for alanylglutamine than for glycylglutamine. Despite this difference, rates of dipeptide and amino acid disappearance during intestinal perfusion were greater from test solutions containing glycylglutamine than alanylglutamine. Furthermore, rates of intraluminal appearance of products of hydrolysis during the infusion of two dipeptides were similar and less than 5% of the disappearance rate of the parent dipeptide. In contrast to free glutamine, uptake of peptide-bound glutamine by brush border membrane vesicles was not inhibited by deletion of sodium or addition of free amino acids to the incubation medium but was inhibited by other oligopeptides and stimulated by a proton gradient. Inhibition constants for the saturable uptake of glycylglutamine and alanylglutamine by vesicles were not significantly different, suggesting similar affinities for the peptide transporter. It is concluded that in human intestine the predominant mechanism for assimilation of glutamine-dipeptides is absorption as intact dipeptide rather than hydrolysis.

Paradoxical Effects of Clofibrate on Liver and Muscle Metabolism in Rats: INDUCTION OF MYOTONIA AND ALTERATION OF FATTY ACID AND GLUCOSE OXIDATION

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.

Evidence for two different modes of tripeptide disappearance in human intestine. Uptake by peptide carrier systems and hydrolysis by peptide hydrolases.

The intestinal fate of two tripeptides (triglycine and trileucine), which differ markedly in solubility and molecular weight, have been investigated by jejunal perfusion in healthy human volunteers. Rates of glycine or leucine uptake from test solutions containing triglycine or trileucine were greater than from test solutions containing corresponding amounts of free glycine or free leucine, respectively. The rate of glycine uptake from a 100 mM triglycine solution was greater than that from a 150 mM diglycine solution. At each infused load of triglycine (e.g., 1,000 mumol/min) the rates (micromoles/minutes per 30 cm) of either triglycine disappearance (810 +/- 40) or glycine absorption (2,208 +/- 122) were markedly greater than the luminal accumulation rates of either diglycine (56 +/- 10) or free glycine (110 +/- 18). The luminal accumulation rate of free leucine during infusion of a 5 mM trileucine solution was over threefold greater than that of free glycine during the infusion of a 5 mM triglycine solution. Luminal fluid exhibited no hydrolytic activity against triglycine, but contained some activity against trileucine. Saturation of free amino acid carrier system with a large load of leucine did not affect glycine absorption rate from a triglycine test solution, but isoleucine markedly inhibited the uptake from a trileucine solution. When the carrier system for dipeptides was saturated with a large amount of glycylleucine, the disappearance rate of triglycine was considerably reduced while that of trileucine remained unaffected. After addition of glycylleucine to tripeptide solutions, there was a minimal increase in the luminal accumulation of diglycine, while dileucine accumulation was incresed by 62-fold.

Fasting-enhanced immune effector mechanisms in obese subjects

Acute nutritional deprivation occurs frequently in clinical practice, yet little data exist on its effect on immune host defenses. To investigate this question, various immune parameters were studied in 15 obese subjects before and after a 14-day fast. Blood monocyte bactericidal activity and natural killer cell cytolytic activity were enhanced by fasting: monocyte killing increased in 12 of 14 subjects (p less than 0.05) and natural killer cell activity increased an average of 24 percent in 13 subjects tested (p less than 0.02). Starvation also enhanced parameters of humoral immunity as evidenced by increases in serum concentrations of IgG, IgA, and IgM (p less than 0.01). By contrast, lymphocyte blastogenic responses to the mitogen phytohemagglutinin were modestly decreased. Peripheral blood leukocyte counts, including neutrophils, T cells, and B cells, did not decrease significantly. These results indicate that fasting has differential influences on immune function rather than a uniformly deleterious effect. Of potential import, this nutritional alteration appears to actually enhance certain effector functions of the host defense system.

Intestinal Absorption of Essential Amino Acids in Man

Summary Equimolar mixtures of the eight essential l-amino acids at varying concentrations (1 to 20 mm) and in amounts usually present in a protein meal were perfused in the jejunum of man and the intestinal absorption kinetics determined. A characteristic sequence and order of amino acid absorption from the mixtures was observed which was constant for each of the amino acids. The absorption rates were most rapid for methionine and the branched amino acids, and slowest for threonine. The succus entericus of the upper jejunum in the fasting state and after perfusion with isotonic saline contains small amounts of amino acids, with leucine and lysine predominating. In an amino acid mixture amino acids may inhibit the absorption of one another if they share a common transport system. The absorption rate of threonine perfused in an equimolar mixture containing the other essential amino acids was markedly decreased compared to its absorption rate when perfused alone in identical concentrations.

The Number of Glycine Residues Which Limits Intact Absorption of Glycine Oligopeptides in Human Jejunum

Studies were performed to determine whether glycine peptides of four or more glycine residues can be transported by the peptide carrier system, previously shown to transport diglycine and triglycine. When human jejunum was perfused with tetraglycine solutions, the rate of tetraglycine disappearance increased linearly as the concentration was increased over the range of 12.5-50 mM, however, the rate was slow in comparison to diglycine and triglycine disappearance rates.Glycylleucine, a competitive inhibitor of diglycine and triglycine transport, was without effect on the disappearance rate of tetraglycine, but increased (over sixfold) appearance rates of triglycine and diglycine (products of tetraglycine hydrolysis). These products were the results of hydrolysis of tetraglycine by the brush border enzymes because cytosol fraction lacked any hydrolase activity against tetraglycine. When a jejunal ring preparation was incubated with tetraglycine, there was intracellular accumulation of diglycine and triglycine but not of tetraglycine. The rates of glycine uptake were always markedly greater from diglycine and triglycine solutions than from corresponding glycine or tetraglycine solutions; rates of glycine uptake from tetraglycine solutions were either similar to or greater than rates from glycine solutions, depending on the infusion concentration. When the number of glycine residues was increased to hexaglycine, the phenomenon of a greater rate of glycine uptake from a peptide versus a free amino acid solution was no longer apparent. In vitro assay of peptide hydrolase activity of the luminal fluid revealed no activity against diglycine and triglycine and only trace activities against tetraglycine, pentaglycine, and hexaglycine. THE ABOVE OBSERVATIONS SUGGEST THE FOLLOWING CONCLUSIONS: (a) the disappearance of tetraglycine in the human jejunum is accomplished principally by hydrolysis by brush border oligopeptidases; (b) the rate limiting step in the uptake of glycine from tetraglycine or higher peptides is due to hydrolysis of these peptides to absorbable products.