Mouillé, Béatrice, Véronique Robert, and François Blachier. Adaptative increase of ornithine production and decrease of ammonia metabolism in rat colonocytes after hyperproteic diet ingestion. Am J Physiol Gastrointest Liver Physiol 287: G344 -G351, 2004. First published April 2, 2004 10.1152/ajpgi.00445.2003.-Chronic highprotein consumption leads to increased concentrations of NH4 ϩ /NH3 in the colon lumen. We asked whether this increase has consequences on colonic epithelial cell metabolism. Rats were fed isocaloric diets containing 20 (P20) or 58% (P58) casein as the protein source for 7 days. NH4 ϩ /NH3 concentration in the colonic lumen and in the colonic vein blood as well as ammonia metabolism by isolated surface colonic epithelial cells was determined. After 2 days of consumption of the P58 diet, marked increases of luminal and colonic vein blood NH4 ϩ /NH3 concentrations were recorded when compared with the values obtained in the P20 group. Colonocytes recovered from the P58 group were characterized at that time and thereafter by an increased capacity for L-ornithine and urea production through arginase (P Ͻ 0.05). L-Ornithine was mostly used in the presence of NH4Cl for the synthesis of the metabolic end product L-citrulline. After 7 days of the P58 diet consumption, however, the ammonia metabolism into Lcitrulline was found lower (P Ͻ 0.01) when compared with the values measured in the colonocytes recovered from the P20 group despite any decrease in the related enzymatic activities (i.e., carbamoylphosphate synthetase I and ornithine carbamoyl transferase). This decrease was found to coincide with a return of blood NH4 ϩ /NH3 concentration in colonic portal blood to values close to the one recorded in the P20 group. In response to increased NH 4 ϩ /NH3 concentration in the colon, the increased capacity of the colonocytes to synthesize L-ornithine is likely to correspond to an elevated Lornithine requirement for the elimination of excessive blood ammonia in the liver urea cycle. Moreover, in the presence of NH 4Cl, colonocytes diminished their synthesis capacity of L-citrulline from Lornithine, allowing a lower cellular utilization of this latter amino acid. These results are discussed in relationship with an adaptative process that would be related to both interorgan metabolism and to the role of the colonic epithelium as a first line of defense toward luminal NH 4 ϩ /NH3 concentrations.

Diallyl disulfide (DADS) is a major organosulphur compound present in garlic with an anti-mitotic potential against colon neoplastic lesions in vivo and colon tumour cell growth in vitro. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line we identified sub-populations of tumoural cells with markedly different characteristics in terms of metabolic capacities, adhesion properties and distribution in the cell cycle phases. After 1 and 2 days treatment with 100 microM DADS HT-29 cells were largely released into the culture medium. These floating cells accumulated in the G(2)/M phase and were characterized by a 5-fold reduction in cell capacity for de novo protein synthesis. Polyamine metabolism, which is necessary for intestinal epithelial cell attachment and growth, was also severely affected, since 3-fold reductions in polyamine biosynthesis and net accumulation of putrescine were measured after DADS treatment. However, oxidation of L-glutamine, the main precursor of the tricarboxylic acid cycle in these cells, and de novo synthesis of glutathione, a tripeptide involved in tumoural cell chemoresistance, were not affected by DADS treatment. In contrast, the adherent sub-population of HT-29 cells, although partially accumulated in G(2)/M phase, were characterized by unaffected metabolic capacities when compared with control cells except for putrescine accumulation, which was transiently decreased, and L-glutamine oxidation, which was increased 2-fold. DADS-resistant cells selected within 5 days were then able to proliferate at a similar rate to control untreated cells. The DADS-induced changes in HT-29 metabolic capacities, adhesion properties and the cell cycle are discussed from a causal perspective.

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Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for the in vitro appraisal of the cytotoxic properties of luminal compounds.