Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription‐polymerase chain reaction (RT‐PCR). The sequence of the RT‐PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT‐PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well‐characterized human and pig colonic lumenal membrane vesicles (LMV). Properties of the colonic lumenal membrane l‐lactate transporter were studied by the uptake of L‐[U‐14C]lactate into human and pig colonic LMV. l‐lactate uptake was stimulated in the presence of an outward‐directed anion gradient at an extravesicular pH of 5.5. Transport of l‐lactate into anion‐loaded colonic LMV appeared to be via a proton‐activated, anion exchange mechanism. l‐lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl− and SO42−. The uptake of l‐lactate was inhibited by phloretin, mercurials and α‐cyano‐4‐hydroxycinnamic acid (4‐CHC), but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid (SITS). The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of l‐lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.

Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [14C]butyrate. The activity of cysteine‐sensitive alkaline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins, markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV were free from contamination by other cellular organelles and basolateral membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein. The transport of butyrate into the luminal membrane vesicles was enhanced 5‐fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperature dependent, and was stimulated in the presence of an outward‐directed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration showed saturation kinetics with an apparent Km value of 14.8 ± 3.6 mM and a Vmax of 54 ± 14 nmol min−1 (mg protein)−1. Butyrate transport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvate and L‐lactate). Butyrate uptake was inhibited by several cysteine group modifying reagents such as p‐chloromercuribenzosulphonic acid (pCMBS), p‐chloromercuribenzoate (pCMB), mersalyl acid and HgCl2, but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonate (DIDS) and 4,4′‐dinitrostilbene‐2,2′‐disulphonate (SITS). The described properties of butyrate transport across the luminal pole of the colon suggest the involvement of a carrier protein, in the form of a pH‐activated anion exchange process. The transporter is distinct from the erythrocyte band‐3 type anion exchanger and may belong to the monocarboxylate‐type transport proteins (MCT1).

Linagliptin administration led to attenuation of postprandial glucose excursions and, despite a low HbA1c at baseline, statistically significant reductions in HbA1c after only 4 weeks of treatment. Linagliptin had a safety and tolerability profile similar to placebo in T2DM patients.

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Regulatory agencies can provide advice to support developers of digital technologies for medicines use, but what are the best strategies to maximize the chance of a successful regulatory interaction? Here, EMA and industry representatives comment on the experience so far.

Liverpool L69 3BX, UK , 'Deparhnent of Gastroenterology, Bndgreen Hosp~tal, Liverpool L14 3LB, LJK , *Clinical Sciences Centre, Nortbem General Hospital Sheffield S5 7AU, UK In many species undigested plant carbohydrates and starch are fermented in the colon by colonic microflora. The major end products of this fermentation are short-chain fatty acids (SCFA) in particular acetate, propionate and butyrate [I].However, other organic compounds such as methane, C a r lactate, and ethanol are also produced in smaller quantities [2]. The significance of the presence of L-lactate nor its transport pathway in the colon have been described before. Whilst hnctional characteristics of L-lactate transport in erythrocytes have been known for many years, the structure of the lactate transporter has only recently been identified at the molecular level and was designated monocarboxylate transporter 1, MCTl, [3]. We set out to investigate the potential presence of a MCTl isoform in pig and human colon by using reverse transcription polymerase chain reaction (RT-PCR) and Northem blot analysis and to determine the plasma membrane location of the protein by Westem blotting.Oligonucleotide primers were designed based on the human heart MCTl cDNA sequence [4] and used in RT-PCR against total RNA isolated from pig and human colonic tissues. The antisense primer, nt 588-611 (5'-GGCCCGATTGGTCGC ATGAGGGCT-3') was used to prime the first strand synthesis and the sense primer, nt 67-90 (S'-GGCTGGGCAGTGGTA ATTGGAGCT-3') was then utilised for DNA amplification. A 544 bp fragment was isolated. The sequence of the RT-PCR product was identical to that of human heart MCT I . Using the 544 bp colonic RT-PCR product as a probe under high stringency conditions, a single transcript of 3.3 kb was observed in RNA isolated from pig and human colonic tissues ( Figure I). The size of this transcript is similar to the reported sizes of MCTl transcripts in rat skeletal muscle [5] and small intestine [6]. The potential presence of a functional MCTl type protein was assessed in purified luminal membrane vesicles (LMV) isolated from pig and human colon [7]. An inward-directed KEN B. HOSE* and SORAYA P. SHIRAZI-BEECHEY A B 3.3 kb 4 W r e 1 : Detection of MCTl bv Northern blot analvsis using the RNA (5 pg per lane) was separated on a denaturing 1% agarose gel containing 0.66 M formaldehyde and transferred to nylon membrane. Hybridisation was carried out at 42°C and washes were performed at 55°C. Lane A, pig colonic RNA; lane B, human colonic RNA.Figure 2: MCTl detection by Western blot analysis in pig & human colonic luminal membrane vesicles Samples (20 pg protein each) were separated on 8% SDS/PAGE. Lane A, pig colonic homogenate; lane B, pig colonic LMV; lane C, human colonic homogenate; lane D, human colonic LMV; lane E, rat erythrocyte ghostproton gradient stimulated the transport of L-lactate 6-fold compared to the absence of a proton gradient. Transport was additionally enhanced by an outward-directed anion gradient. Transport was inhibited by pyruvate a...

The main source of nitrogen (N) in the colon is ammonia derived from urea [I]. In mammals, urea N plays an important source of N during low protein intake [2]. Ruminants use recycled N in order to maintain microbial growth in the reticulorumen and hindgut [I]. Urea transporters (UT) have been identified in rat [3, 41, rabbit [5] and human kidney [6], and human bone marrow cells [7] and corresponding cDNAs cloned and sequenced. Recently, the rat equivalent to the human erythrocyte urea transporter cDNA (HUT1 1) was cloned and designated UT3 [4]. All three isofonns of the rat UT are expressed in the kidney [4].In our previous studies we investigated the mechanism for the transport of urea across sheep rumen and colonic epithelia. Our data suggested the presence of a carrier mediated, facilitated transport mechanism for urea across the epithelial cells of these tissues [8]. Northern blot analysis using the full length rabbit UT2 cDNA did not produce a signal with RNA isolated from colonic and rumen tissues [8]. A weak signal was detected in human colonic tissue using the HUT1 1 cDNA [6]. These findings raised the question of whether the UT in the human colon was structurally more closely related to HUT1 1 than HUTZ. Based on these data, we designed oligonucleotide primers against the human erythrocyte UT, HUT1 I, for use in reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR reaction was performed in a single tube containing 1 pg total RNA isolated from human colon, sheep rumen or rat kidney.The antisense primer, nt 787-802 (5'-GCAGCATGCAGGCACATGAG-3') was used for the reverse transcription and the sense primer, nt 211-230 ( 5 ' -GGCATATCCCAAGTGGTGTT-3') was used for DNA amplification. Figure 1 : Analysis of RT-PCR products 1 pI of the RT-PCR reaction mixture was analysed on a 1.6 % agarose gel.The desired amplicons were directly subcloned into pGEM-T vector (Promega) and custom sequenced (University of Durham). SpeciesIdentity (%) human colon ShCW lumen human colon Fragment 100 sheep rumen fragment 87 rat kidney fragment 95.8 human erythrocyte UT (HUT1 1) 81.7 human kidney UT (HUT2) 70.9 rat kidney UT (UT3) 95.8 rabbit kidney UT (UT2) 72.4 rat kidney UT (rUT2) 70.5 87 100 87 81.5 68.8 87.2 70.1 69.5 ~~ "&le 1. C-son of nucleotide Folon and sheeo rumen RT-PCR fragments with & i transM)rters Comparisons are based on sequenced human colonic, sheep rumen and rat kidney RT-PCR fragments using LALIGN.Using primers against the human erythrocyte UT (HUTI1) in RT-PCR, we identified products of similar sizes in RNA samples isolated from rat kidney, human colon and sheep rumen (Figure 1). RNA isolated from rat kidney was used as a positive control for RT-PCR. Sequencing of the purified pmducts nvwled close similarities amongst the three. As expected, the rat kidney fragment was identical to rat UT3.In Table 1 the nucleotide sequences of the cloned human colon and sheep rumen RT-PCR products have been compared with other urea transport proteins identified to date. Interestingly, the sequence of the human colo...