Volume 116, Issue 2, Pages 354-362 (February 1999) Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1  Toshiyuki Shiraga, Ken-Ichi Miyamoto, Hiroko Tanaka, Hironori Yamamoto, Yutaka Taketani, Kyoko Morita, Ikumi Tamai, Akira Tsuji, Eiji Takeda  Gastroenterology  Volume 116, Issue 2, Pages 354-362 (February 1999) DOI: 10.1016/S0016-5085(99)70132-0 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Time courses of Gly-Sar uptake into BBMVs from rats fed a protein-free or a 50% casein diet. BBMVs from rats fed the nonprotein diet and rats fed the casein diet (5%, 20%, and 50%) were preloaded with 280 mmol/L mannitol and 20 mmol/L HEPES-Tris (pH 8.4) and then incubated for the indicated times at 20°C in the presence of 100 mmol/L NaCl, 80 mmol/L mannitol, 20 mmol/L HEPES-(2-[N-morpholino]ethanesulfonic acid) (MES) (pH 5.5), and 1 mmol/L [3H]Gly-Sar. Data are means ± SE of 6 rats. **P < 0.01, *P < 0.05 vs. protein-free diet. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of dietary protein on the expression of PepT1 in the rat ileum. (A) Northern blot analysis was performed on total RNA (20 μg) from the intestine. (B) Immunoblot analysis of PepT1 in the rat small intestine. PepT1 immunoreactivity was determined in BBMVs from the ileum. Each lane contained 50 μg of protein. Lane 1, protein-free diet; lane 2, 5% casein diet; lane 3, 50% casein diet. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of dipeptide diet (20% Gly-Phe) and single amino acid (10% Gly or 10% Phe) diet on Gly-Sar uptake and PepT1 expression in the rat small intestine. (A) BBMVs were isolated from rats after 3 days of the diets, and the transport activity was analyzed. (B) A Northern blot analysis was performed on total RNA (20 μg) from the intestine. (C) The relative concentrations of PepT1 protein (Western blotting) were determined by densitometric scanning of autoradiographs. Results are the means ± SE of the relative percent of the protein-free diet group. *P < 0.01 vs. protein-free diet. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Nucleotide sequence of the rat PepT1 gene. The large boxed sequence indicates the transcribed nucleotides (exon 1), and the translational start codon of the rat PepT1 protein is underlined. The major transcriptional start site as determined by the primer extension and S1 nuclease assays is indicated by the arrow. The TATA and GC box sequences are indicated by the small boxes. Several potential sites for various transacting factors are overlined. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Transient transfection experiments using rat PepT1 promoter-luciferase deletional constructs. Deletional constructs of the rat PepT1 gene promoter region were linked to the luciferase reporter gene as described in Materials and Methods. The pCMV β-gal plasmid, which contains the lacZ gene linked to the cytomegalovirus promoter and enhancer, was used as a control for transfection efficiency. Caco-2 (■), HepG2 (▨), and opossum kidney (□) cells were cotransfected with RPT-LUC construct (0.8 μg) and pCMV β-gal (0.2 μg). Controls were performed in each experiment, including transfection with pGL3-basic vector (promoter and enhancerless). The luciferase activity values were normalized for transfection efficiency using the β-galactosidase activity, and the data are expressed as light units/unit of β-galactosidase activity. Data are mean ± SE of 3 independent transfections. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of a dipeptide and a single amino acid on Caco-2 cells transfected with rat PepT1 promoter-luciferase constructs. (A) Caco-2 cells were transfected with −50, −97, −171, −351, or −1.6-k RPT-LUC deletion clone. After transfection, the cells were cultured in DMEM containing 10 mmol/L Gly-Phe (▨) or 20 mmol/L Phe (■) for 48 hours. Control experiments (□) were performed by using standard DMEM. The data are mean ± SE of 3 independent transfections. (B) Caco-2 cells were transfected with −351 RPT-LUC clone. After transfection, the cells were cultured in DMEM containing 10 or 20 mmol/L of various dipeptides and amino acids for 48 hours. The substrates added for each experiment were 20 mmol/L Gly-Sar, 10 mmol/L Gly-Phe, Phe-Val, Lys-Phe, or Asp-Lys, and 20 mmol/L Gly, Val, Phe, Lys, Asp, Gln, Arg, or Ala. Control experiments (non) were performed by using standard DMEM. The values of luciferase activity, expressed as a fold increase of the −351 RPT-LUC activity in DMEM medium, are on the bottom. The data are mean ± SE of 5 independent transfections. *P < 0.05 vs. control. **P < 0.01 vs. control. Gastroenterology 1999 116, 354-362DOI: (10.1016/S0016-5085(99)70132-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions