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.

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.