Absorption of small peptides makes a significant contribution to total dietary protein assimilation (Grimble & Silk, 1989; Matthews, 1991). cDNAs for rabbit (Fei et al. 1994), human (Liang et al. 1995) and rat (Saito, Okuda, Terada, Sasaki & Inui, 1995) intestinal, proton-coupled peptide transporters (PepT1, hPepT1 and rat PepT1, respectively) have been cloned. As suggested by earlier functional studies (Thwaites, Brown, Hirst & Simmons, 1993a; Yuasa, Fleisher & Amidon, 1994), these cDNAs confer proton-coupled absorption in Xenopus laevis oocytes of a number of clinically important drugs, including β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, as well as of di- and tripeptides (Boll, Markovich, Weber, Korte, Daniel & Murer, 1994; Fei et al. 1994). Regulation of hPepT1, therefore, has both nutritional and therapeutic implications.
Substrate upregulation of nutrient transporters is a mechanism for ensuring economy to the cell of synthetic and maintenance costs. It is well exemplified by the Na+-glucose cotransporter, SGLT1, which is upregulated in rats and mice by a high carbohydrate diet (Diamond, Karasov, Cary, Enders & Yung, 1984; Cheeseman & Harley, 1991; Ferraris, Villenas, Hirayama & Diamond, 1992), and in sheep by infusion of glucose into the intestine (Lescale-Maty, Dyer, Scott, Wright & Shirazi-Beechey, 1993). Regulation of the ovine transporter is principally by translational or post-translational mechanisms. Additionally, in rat there is evidence for a rapid regulatory response of SGLT1 to jejunal glucose that precedes the onset of increased protein expression at the lumenal membrane (Sharp, Debnam & Srai, 1996). In the human intestine the abundance and activity of SGLT1 is maintained by lumenal nutrients (Dyer, Hosie & Shirazi-Beechey, 1997). In vivo feeding studies in rats and mice suggest that the proton-coupled peptide transporter also demonstrates this form of regulation. Uptake of the dipeptide L-carnosine into everted intestinal sleeves of mice fed a high (72 %) protein diet compared with a low (18 %) protein diet was increased (30–70 %) in proximal regions of the gut (Ferraris, Diamond & Kwan, 1988). In rats, a switch from a low protein diet, comprising 4 % casein, to a high protein diet, containing 50 % gelatine, produced increases in PepT1 mRNA of 1.5- to 2-fold (Erickson, Gum, Lindstrom, McKean & Kim, 1995). However, the precise inducer(s) of functional PepT1 expression remains to be established, and the mechanism by which the increase in PepT1 accumulation occurs has not yet been investigated. In addition, it is unclear whether the dietary effect on PepT1 is elicited via a process acting directly on small intestinal enterocytes or through indirect hormonal and/or neural pathways. These important questions are not readily amenable to resolution by studies of peptide transport in the whole animal. In vitro, cell line models offer a more appropriate system for investigation of the control of PepT1 expression. They afford the opportunity to manipulate precisely and easily nutrient levels in the culture medium and are more accessible than in vivo models to examination of the regulatory mechanism at the molecular level. The Caco-2 cell line is derived from a human colon adenocarcinoma and grows as an adherent monolayer of differentiated, polarized cells that show many features of the typical small intestinal enterocyte (Hidalgo, Raub & Borchardt, 1989). Correct polarity of expression in Caco-2 cells of a number of proteins, for example lactase, sucrase-isomaltase (Van Beers, Al, Rings, Einerhand, Dekker & Buller, 1995) and neutral endopeptidase (Jalal et al. 1992), has been demonstrated. The cell line is a well-established model for the functional study of proton-coupled dipeptide uptake and transepithelial transport (Thwaites, Brown, Hirst & Simmons, 1993b; Brandsch, Miyamoto, Ganapathy & Leibach, 1994).
The present study utilizes the Caco-2 cell line to address the question of the molecular mechanism of peptide transport regulation by a substrate. The data demonstrate that hPepT1 activity, protein and mRNA levels in Caco-2 cells are upregulated by the dipeptide Gly-Gln. Increased hPepT1 mRNA levels in response to substrate are a reflection of both increased transcription and mRNA stability mediated via a signalling pathway acting directly on the enterocyte.
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A physiological response to increased levels of dietary protein (Ferraris et al. 1988; Erickson et al. 1995) has been confirmed in human intestinal Caco-2 cells in vitro, as increased functional activity of apical dipeptide (Gly-Sar) and amino acid uptake, indicating that this is a direct effect of nutrient supply on epithelial cell function, without a requirement for neural and/or hormonal mediators. Gly-Sar, a non-hydrolysable dipeptide, is a substrate of the cloned proton-coupled peptide transporter hPepT1 (Liang et al. 1995) and increased upake, therefore, is consistent with increased hPepT1 activity. The underlying molecular mechanism for this upregulation of functional activity is increased levels of hPepT1 mRNA and concomitant increases in hPepT1 protein expression. The magnitude of the increases in hPepT1 activity, mRNA and protein levels is in the range 1.64-1.92 times basal values, demonstrating that control of mRNA accumulation, rather than translation or post-translational modification, is the primary mechanism of regulation. Furthermore, the close agreement between the enhanced levels of dipeptide uptake (1.64-fold) and hPepT1 protein expression (1.72-fold) shows that hPepT1 alone accounts for all peptide-regulated Gly-Sar uptake into Caco-2 cells.
An increase in hPepT1 mRNA stability accounts, at least to some degree, for the observed upregulation, with half-life increasing from 8.9 to 12.5 h. A discrepancy between the calculated level of hPepT1 mRNA accumulation, resulting from a change of this magnitude, and the measured increase in hPepT1 mRNA indicates that there is also a contribution from increased hPepT1 mRNA transcription. Direct demonstration of a transcriptional component to the regulatory response to peptide, by nuclear run-off analysis, was precluded by the very low levels of hPepT1 mRNA expression and, therefore, awaits examination of control and peptide-induced levels of expression by other, more sensitive, techniques, such as the use of a reporter gene coupled to the hPepT1 promoter region.
Increased hPepT1 mRNA stability could, speculatively, arise from reduced expression of a message-destabilizing factor, increased expression of a message-stabilizing factor or altered interactions between hPepT1 mRNA and such factors. A number of recent studies report the involvement of mRNA-binding protein factors in the regulation of mRNA stability (e.g. Zhang et al. 1993; Preiss, Sang, Chrzanowska & Lightowlers, 1995; Geneste, Raffalli & Lang, 1996; Pende et al. 1996). Involvement of intracellular second messenger signalling molecules in the modulation of mRNA stability has also been reported (reviewed in Williams, Sensel, McTigue & Binder, 1993). For the Na+-coupled glucose transporter, SGLT1, for example, elevated cAMP resulted in increased message stability (Peng & Lever, 1995), and protein kinase C activation has been implicated in message destabilization (Shioda, Ohta, Isselbacher & Rhoads, 1994). Downregulation of hPepT1 by both cAMP (Muller, Brandsch, Prasad, Fei, Ganapathy & Leibach, 1996) and protein kinase C (Brandsch et al. 1994) has been reported, although the fact that these responses are observed over a time scale as short as 2 h, plus the fact that the protein kinase C-mediated decrease in hPepT1 activity is independent of de novo protein synthesis (Brandsch et al. 1994), suggests that they are distinct from the upregulation reported here. Nonetheless, this does not rule out a role for protein kinase A and/or protein kinase C in the longer-term response to Gly-Gln.
Under our standard experimental conditions, the signal for hPepT1 upregulation might be the dipeptide itself or changes in levels of the constituent amino acids Gly and Gln in the culture medium. This issue is addressed by the observation that even under conditions where complete extracellular hydrolysis of added dipeptide would have produced no net increase in amino acid concentration in the nutrient medium (addition of Gly-Gln reduced to 2 mM, whilst glutamine was still depleted by 4 mM), upregulation of hPepT1 mRNA levels to the same degree as observed under our standard conditions was still measured (Picker, Hall, Walker & Hirst, 1997). This indicates that extracellular hydrolysis of dipeptide to increase net amino acid concentration is not required to induce transporter upregulation. However, this does not exclude a role for apical peptidase activity in the signalling mechanism for hPepT1 upregulation. It is also impossible, at this stage, to rule out the possibility of intracellular hydrolysis being necessary to induce transporter upregulation. In fact, our choice of the dipeptide Gly-Gln to induce hPepT1 upregulation was based on it being an hPepT1 substrate which, whilst being relatively robust, is still hydrolysed by intestinal brush-border membrane hydrolases to some degree (Minami, Morse & Adibi, 1992) to release the component, metabolically active amino acids, of which Gln in particular is an important energy source for the gut (Ashy, Salleh & Ardawi, 1988). By satisfying these criteria, Gly-Gln represents a physiological dietary product whose assimilation is mediated by hPepT1 and which provides the intestinal cells, and the organism in general, with important nutrients.
The response of PepT1 mRNA levels to a protein-rich diet in rats (Erickson et al. 1995) could also be dipeptide mediated. However, an earlier study which found uptake of the non-hydrolysable dipeptide L-carnosine into everted mouse intestinal sleeves to be increased by elevated levels of dietary protein (casein) also showed that, at an intermediate level of dietary protein, uptake levels were independent of its composition, with full hydrolysate producing the same effect as non-hydrolysed casein (Ferraris et al. 1988). It seems possible, then, that at least two different pathways might contribute to adaptive upregulation of PepT1.
A further pertinent question is whether the parallel response of amino acid transport systems, demonstrated here as increased Lys and Leu uptake, is mediated via a dipeptide-stimulated pathway, or if hydrolysis to amino acids is required first. This, and the other issues concerning the nature of the regulatory stimulus discussed above, will be the subject of further investigation.
Concerning the systemic link between protein/dipeptide in the gut lumen and the regulatory response, our study has shown that dipeptide-mediated upregulation of PepT1 in the enterocyte involves a direct effect that operates independently of hormonal and/or neural pathways, and that these effects can account for the full magnitude of upregulation reported in vivo (Ferraris et al. 1988; Erickson et al. 1995).
Evidence for the existence of discrete or common peptide transport mechanisms in the apical and basolateral membranes of the enterocyte is equivocal. Different profiles for pH dependency of Gly-Sar (Thwaites et al. 1993b) and bestatin (Saito & Inui, 1993) uptake into Caco-2 cells across apical and basolateral surfaces, plus selective inhibition by p-chloromercuribenzene sulphonate at apical and basolateral Caco-2 membranes (Saito & Inui, 1993) point to discrete transporters. The results of hybrid depeletion studies, indicating PepT1 accounts for all Gly-Sar uptake activity in Xenopus laevis oocytes injected with rabbit small intestinal mRNA, are, however, apparently in conflict with these data (Fei et al. 1994). By use of hPepT1-specific antibodies, we have immunolocalized hPepT1 expression exclusively to the apical membranes of human enterocytes in vivo and Caco-2 cells in vitro. This provides further evidence for a distinct basolateral peptide transporter.
In conclusion, we have demonstrated that Caco-2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. Levels of hPepT1 mRNA and protein are increased by an amount commensurate with the magnitude of functional increase in activity in response to Gly-Gln. The level of hPepT1 upregulation demonstrated in this epithelial model represents the full, in vivo response. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte and operates independently of hormonal and/or neural control. Upregulation is mediated in part via increased hPepT1 mRNA stability. A role for increased hPepT1 gene transcription is also likely: however, establishing this unequivocally awaits additional studies using the hPepT1 promoter region coupled to a sensitive reporter gene. Further clarification of the molecular details of this regulatory response requires the demonstration and identification of putative hPepT1 mRNA-interacting proteins and investigation of the involvement of recognized intracellular second messenger signalling pathways.