Substrate upregulation of the human small intestinal peptide transporter, hPepT1.
Walker D. Thwaites DT. Simmons NL. Gilbert HJ. Hirst BH.
Department of Physiological Sciences, University of Newcastle upon Tyne, Newcastle Upon Tyne NE2 4HH, UK. Dianne.Walker@ncl.ac.uk
1. Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl-L-glutamine (Gly-Gln) of the proton-coupled peptide transporter, hPepT1, in the Caco-2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl-[14C]sarcosine was increased 1.64 (+/- 0.34)-fold after incubation of Caco-2 cells for 3 days in a peptide-rich medium (4 mM Gly-Gln replacing 4 mM Gln). 2. A full-length Caco-2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile-662 to Val. Transcript sizes, on Northern blots of Caco-2 poly(A)+ RNA probed with a 630 bp 5' hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide-induced increase in substrate transport was accompanied by a parallel increase of 1.92 (+/- 0.30)-fold (n = 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half-life from 8.9 +/- 1.1 to 12.5 +/- 1.6 h (n = 3), but the increase in half-life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide-mediated increase in hPepT1 transcription. 3. Anti-hPepT1-specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco-2 cells. Gly-Gln supplementation of media resulted in a 1.72 (+/- 0.26)-fold (n = 5) increase in staining intensity of Caco-2 cells. 4. We conclude 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. The magnitude of functional increase in apical peptide transport activity in response to Gly-Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.