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.

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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-3 HGln 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 K m 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.Lysosomes are major sites for protein degradation in the liver. Amino acids and dipeptides are the final products of proteolysis within this organelle (1, 2). These products must be exported into the cytosol if lysosomes are to retain their biological integrity. Failure to export may result in pathological consequences. This is evidenced by the genetic defect in the export of cystine, which results in accumulation of a high concentration of this amino acid in the lysosomes causing cystinosis. Cystinosis is a progressive systemic disease with many pathological expressions (3).Although as yet no genetic disease of dipeptide accumulation in lysosomes has been described, there is evidence that such an accumulation would also have pathological effects. This is evidenced by the osmotic protection experiments which determine the rates of release of lysosomal enzymes. Lloyd (4) showed that incubation of lysosomes isolated from rat liver with 0.25 M solutions of dipeptides or their constituent amino acids increased the release of their enzyme contents. Furthermore, the release was greater with dipeptides than with their constituent amino acids. He suggested that amino acids and dipeptides enter the lysosomes, and the entry is more efficient for dipeptides than for amino acids. Recently, Bird and Lloyd (5) found that the rate of enzyme release was greater with dipeptides containing L-isomers than D-analogs, suggesting a stereospecificity in lysosomal permeation of dipeptides. However, as reviewed by Forster and Lloyd (6), the osmotic protection technique does not allow any conclusion regarding the mechanism which a solute uses for entry into the lysosomes. For example, the greater rate of enzyme release by dipeptides containing Lrather than D-amino acids could be explained by the hydrolysis of dipeptide...