Effects of the L-tyrosine-derived bacterial metabolite p-cresol on colonic and peripheral cells.

Specific families of bacteria present within the intestinal luminal content produce p-cresol from L-tyrosine. Although the hosts do not synthesize p-cresol, they can metabolize this compound within their colonic mucosa and liver leading to the production of co-metabolites including p-cresyl sulfate (p-CS) and p-cresyl glucuronide (p-CG). p-Cresol and its co-metabolites are recovered in the circulation mainly conjugated to albumin, but also in their free forms that are excreted in the urine. An increased dietary protein intake raises the amount of p-cresol recovered in the feces and urine, while fecal excretion of p-cresol is diminished by a diet containing undigestible polysaccharides. p-Cresol in excess is genotoxic for colonocytes. In addition, in these cells, this bacterial metabolite decreases mitochondrial oxygen consumption, while increasing the anion superoxide production. In chronic kidney disease (CKD), marked accumulation of p-cresol and p-CS in plasma is measured, and in renal tubular cells, p-cresol and p-CS increase oxidative stress, affect mitochondrial function, and lead to cell death, strongly suggesting that these 2 compounds act as uremic toxins that aggravate CKD progression. p-Cresol and p-CS are also suspected to play a role in the CKD-associated adverse cardiovascular events, since they affect endothelial cell proliferation and migration, decrease the capacity of endothelial wound repair, and increase the senescence of endothelial cells. Finally, the fact that concentration of p-cresol is transiently increased in young autistic children biological fluids, and that intraperitoneal injection of p-cresol in animal models induces some behavioral characteristics observed in the autism spectrum disorders (ASD), raise the view that p-cresol may possibly represent one of the components involved in ASD etiology. Further pre-clinical and clinical studies are obviously needed to determine if the lowering of p-cresol and/or p-CS circulating concentrations, by dietary and/or pharmacological means, would allow, by itself or in combination with other interventions, to improve CKD progression and associated cardiovascular outcomes, as well as some neurological outcomes in children with an early diagnosis of autism.

Dietary supplementation with Bacillus mixture modifies the intestinal ecosystem of weaned piglets in an overall beneficial way.

AIMS: This study was conducted to investigate the effects of dietary supplementation with a mixture of Bacillus, which serves as an alternative of antibiotics on the intestinal ecosystem of weaned piglets. METHODS AND RESULTS: We randomly assigned 120 piglets to three groups: a control group (a basal diet), a probiotics group (a basal diet supplemented with 4 × 109  CFU per gram Bacillus licheniformis-Bacillus subtilis mixture; BLS mix), and an antibiotics group (a basal diet supplemented with 0·04 kg t-1 virginiamycin, 0·2 kg t-1 colistin and 3000 mg kg-1 zinc oxide). All groups had five replicates with eight piglets per replicate. On days 7, 21 and 42 of the trial, intestine tissue and digesta samples were collected to determine intestinal morphology, gut microbiota and bacterial metabolite composition, and the expression of genes related to the gut barrier function and inflammatory status. The results showed that the BLS mix decreased the jejunum crypt depth, while increased the ileum villus height and the jejunum and ileum villus height to crypt depth ratio. The BLS mix increased Simpson's diversity index in the gut microbiota and the relative abundances of o_Bacteroidetes and f_Ruminococcaceae, but decreased the relative abundances of Blautia and Clostridium. Dietary BLS mix supplementation also modified the concentration of several bacterial metabolites compared to the control group. In addition, BLS mix upregulated the expression level of E-cadherin in the colon and pro-inflammatory cytokines and TLR-4 in ileum and colon. Lastly, Spearman's rank-order correlation revealed a potential link between alterations in gut microbiota and health parameters of the weaned piglets. CONCLUSION: These findings suggest that dietary BLS mix supplementation modifies the gut ecosystem in weaned piglets. The potential advantages of such modifications in terms of intestinal health are discussed. SIGNIFICANCE AND IMPACT OF THE STUDY: Weaning is the most important transition period of piglet growth and development. This study showed that dietary supplementation of a probiotic mixture of Bacillus, an effective alternative of antibiotics, was beneficial in improving the intestinal ecosystem of weaned piglets.

Stages of pregnancy and weaning influence the gut microbiota diversity and function in sows.

AIMS: The gut microbiota is believed to play important roles in the health of pregnant mammals, including their nutrient metabolism, immune programming and metabolic regulation. However, until recently, the shifts in gut microbiota composition and faecal and blood metabolic activity during different stages of pregnancy had not been investigated. METHODS AND RESULTS: We investigated the shifts in backfat thickness, plasma and faecal metabolites and gut microbiota on days 30, 60, 90 and 110 of pregnancy and on day 21 after parturition (weaning) in sows. The backfat thickness of sows did not significantly differ among the different stages of pregnancy. The plasma concentrations of lipid metabolites, including triacylglycerol (TG), total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol and calcium were reduced (P < 0·05) during pregnancy. In addition, the concentration of these metabolites, except TG, reached their maximum at the time of weaning. We also found that Tenericutes, Fibrobacteres and Cyanobacteria varied significantly according to the stages of pregnancy in sows (P < 0·05). Most of the genera, such as Clostridiales, Desulfovibrio, Mogibacteriaceae and Prevotella, increased (P < 0·05) with the progression of pregnancy and decreased (P < 0·05) at weaning. The alpha diversity values (i.e., Shannon diversity and observed species) of sow gut microbiota increased (P < 0·05) from pregnancy to weaning. Pregnancy stages also significantly influenced (P < 0·05) the community structure (beta diversity) of gut microbiota. The progression of pregnancy was associated with changes in lipid metabolism and several carbohydrate-degradation bacteria (i.e., Prevotella, Succinivibrio, Bacteroides and Parabacteroides). CONCLUSIONS: Although causal links between the measured parameters remain hypothetical, these findings suggest that the increased diversity and concentration of beneficial gut microbes are associated with the metabolism of pregnant sows. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulation of the sow gut microbiota composition may potentially influence metabolism and health during pregnancy.

Colonic luminal microbiota and bacterial metabolite composition in pregnant Huanjiang mini-pigs: effects of food composition at different times of pregnancy.

The gut harbours diverse and complex microbiota, which influence body health including nutrient metabolism, immune development, and protection from pathogens. Pregnancy is associated with immune and metabolic changes that might be related to microbiota compositional dynamics. We therefore investigated the colonic luminal bacteria community in Huanjiang mini-pigs fed diets with different nutrient levels from the first to third trimester of pregnancy. The concentrations of intestinal metabolites including short-chain fat acids, NH3-N, indole, skatole, and bioamines were also determined. We found that the colonic bacteria species richness estimators (Chao1 and ACE) decreased with increased gestational age. The dominant phyla identified were Firmicutes and Bacteroidetes; the dominant genera were Lactobacillus, Treponema, Ruminococcus, Clostridium, and Prevotella. In addition, microbiota displayed spatial and temporal heterogeneity in composition, diversity, and species abundance in different colonic segments from the first to third trimester of pregnancy. Furthermore, the bacterial metabolites also changed according to the diet used and the pregnancy stage. These findings suggest that colonic bacteria richness decreased as gestational age increased, and that the higher nutrient level diet increased the production of metabolites related to nitrogen metabolism. However, although the higher nutrient diet was associated with pregnancy syndrome, causal links remain to be determined.

Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle.

A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner.

Effects of amino acid-derived luminal metabolites on the colonic epithelium and physiopathological consequences.

Depending on the amount of alimentary proteins, between 6 and 18 g nitrogenous material per day enter the large intestine lumen through the ileocaecal junction. This material is used as substrates by the flora resulting eventually in the presence of a complex mixture of metabolites including ammonia, hydrogen sulfide, short and branched-chain fatty acids, amines; phenolic, indolic and N-nitroso compounds. The beneficial versus deleterious effects of these compounds on the colonic epithelium depend on parameters such as their luminal concentrations, the duration of the colonic stasis, the detoxication capacity of epithelial cells in response to increase of metabolite concentrations, the cellular metabolic utilization of these metabolites as well as their effects on colonocyte intermediary and oxidative metabolism. Furthermore, the effects of metabolites on electrolyte movements through the colonic epithelium must as well be taken into consideration for such an evaluation. The situation is further complicated by the fact that other non-nitrogenous compounds are believed to interfere with these various phenomenons. Finally, the pathological consequences of the presence of excessive concentrations of these compounds are related to the short- and, most important, long-term effects of these compounds on the rapid colonic epithelium renewing and homeostasis.

Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells.

Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.

Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: effects of hydrogen sulfide, ammonia, and deoxycholic acid.

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.

Effects of the garlic compound diallyl disulfide on the metabolism, adherence and cell cycle of HT-29 colon carcinoma cells: evidence of sensitive and resistant sub-populations.

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.

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.

Inducible nitric oxide synthase activity in colon biopsies from inflammatory areas: correlation with inflammation intensity in patients with ulcerative colitis but not with Crohn's disease.

Nitric oxide synthase (NOS) activities are responsible for the enzymatic conversion of L-arginine into NO and L-citrulline. Relatively low amounts of NO are produced in intestinal epithelial cells or are released from nerve endings. The effects of NO production are related to the maintenance of epithelial integrity and permeability. A pathological role of an increased NO production has been suggested to play a role in models of experimental colitis. In humans, NOS activity in colon mucosa from patients with ulcerative colitis is clearly increased when compared with the activity of the control group. In contrast, an increase of NOS activity in the colon mucosa from patients with Crohn's disease remains controversial. In the present work, we have measured NOS activity in colon biopsies originating from the control group (n = 16), from patients with ulcerative colitis (n = 23) and Crohn's disease (n = 17) using the radiochemical method of the conversion of L-[guanido-14C] arginine into radioactive L-citrulline. In the control group, NOS activity was mainly of the inducible type (88% of total NOS activity) since it was characterised by its insensibility to the absence of calcium in the assay medium. In colon biopsies originating from patients with ulcerative colitis, inducible NOS activity was increased 3 fold (p < 0.005) and in patients with Crohn's disease, inducible NOS activity was increased 5 fold (p < 0.005). Correlations between NOS activity in colon biopsies and the intensity parameters of the disease i.e. Truelove index, endoscopic score and histological parameters were evidenced in patients with ulcerative colitis. In contrast, in patients with Crohn's disease, the high inducible NOS activity was not correlated with any intensity parameters of the disease. From these data, we concluded that although inducible NOS activity was increased several fold in colon biopsies originating from patients with both ulcerative colitis and Crohn's disease, a correlation between this activity and the severity of bowel inflammation was not found in either cases.

Portal hyperglutamatemia after dietary supplementation with monosodium glutamate in pigs.

The aim of the present work was to examine in pigs the effect of a dietary supplementation with the flavor enhancer monosodium glutamate (MSG) on intestinal amino acid metabolism. For this purpose, pigs weighing 60 +/- 2 kg received a standard meal twice a day for 1 week, supplemented with either 10 g MSG per meal or, as control experiments, an isonitrogenous amount of glycine together with an equal amount of sodium in the form of NaCl, the animals being their own control in all experiments. At the end of this period, pigs received a MSG or glycine-NaCl-supplemented meal and samples of portal and arterial blood were collected for amino acid analysis in plasma. The results demonstrate after MSG supplementation rapid significant increases in glutamate concentration in the portal and arterial blood plasma after a test meal which resulted in a positive portoarterial difference. In comparison, after glycine-NaCl supplementation, glutamate concentrations were almost identical in portal and arterial plasma. Furthermore, significant increased aspartate concentration in the portal blood plasma was observed after MSG supplementation when compared with control experiments. When enterocytes were isolated at the end of the supplementation period from the jejunum and examined for their metabolic capacities towards L-glutamate and L-glutamine, it was found that metabolism did not differ according to the supplement used, with glutamate and glutamine being oxidized and transaminated at a similar level. It is concluded that the portal hyperglutamatemia observed shortly after the ingestion of a MSG- supplemented meal is likely due to the saturation of the intestinal capacity to metabolize glutamate with no measurable adaptation of the metabolic pathways controlling glutamate metabolism in enterocytes.

Metabolic capacity for L-citrulline synthesis from ammonia in rat isolated colonocytes.

Ammonia is present at high concentration in the colon lumen and is considered a colon cancer suspect. Furthermore, ammonia usually eliminated by the liver in the ornithine cycle is considered highly toxic to cerebral function when present in excess in the blood plasma. Therefore, the metabolic pathways involved in ammonia metabolism in colonocytes were studied in the present study. Rat colonocytes were found equipped with low carbamoylphosphate synthase I activity, high ornithine carbamoyltransferase and arginase activities and low argininosuccinate synthase activity. High (10 and 50 mmol/l) NH4Cl concentrations but not low concentrations (1 and 5 mmol/l) were found able to increase respectively 3- and 10-fold the conversion of radioactive L-arginine to L-citrulline. In contrast, very low capacity for L-citrulline conversion to L-arginine is found in colonocytes. It is concluded that an incomplete ornithine cycle is operative in colonocytes which results in ammonia stimulated L-citrulline production. The contribution of this metabolic pathway in relation to ammonia detoxication by colonocytes is discussed.

Can a glutamate-enriched diet counteract glutamine depletion in endotoxemic rats?

The study evaluated whether a glutamate-enriched diet would restore glutamine tissue pools and maintain tissue trophicity in endotoxemic rats. For this purpose, young male Sprague-Dawley rats received an intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli at 3 mg/kg body weight. After 24 hours of food deprivation, the rats were enterally refed for 48 hours using Osmolite enriched with glutamate at 4 g/kg/d (LPS-Glu group, n = 7) or glycine isonitrogenous to glutamate (LPS-Gly group, n = 7). A control group (healthy group, n = 7) had free access to a standard rodent diet. Tissue weights and protein contents were significantly lower in both LPS-treated groups than in the healthy group. No plasma or tissue accumulation of glutamate was observed except in the liver. Glutamine concentrations were increased in the jejunum, liver, and plasma in the LPS-Glu group versus the other two groups (P < 0.05). Conversely, they were depleted in muscles of the endotoxemic groups versus the healthy group (P < 0.05). Villus height was significantly greater in the LPS-Glu group than in the LPS-Gly group in the jejunum (P < 0.05), but not in the ileum. In conclusion, a glutamate-enriched diet administered enterally to endotoxemic rats can counteract glutamine depletion in the splanchnic area but not in muscles. In addition, glutamate displayed a trophic effect restricted to the jejunum.

De novo synthesis of arginine and ornithine from citrulline in human colon carcinoma cells: metabolic fate of L-ornithine.

In human colon carcinoma cells (HT-29 cells), L-arginine is the common precursor of L-ornithine which generates polyamines strictly necessary for cellular growth, and nitric oxide which has a strong antiproliferative activity. We show here that proliferative HT-29 cells possess the capacity for de novo synthesis of L-arginine from L-citrulline, but not from L-ornithine. L-Ornithine is apparently not an L-arginine precursor due to the absence of any detectable ornithine carbamoyltransferase activity. In contrast, the newly synthesized L-arginine was competent for urea and thus L-ornithine production in a context of a high putrescine production in the ornithine decarboxylase pathway and a low degradation of this polyamine in the diamine oxidase pathway. However, cells grown in an arginine-free culture medium containing added L-citrulline were unable to reach confluency. Furthermore, the low amount of nitric oxide produced from L-arginine by these cells was apparently not involved in the control of cell growth since inhibition of nitric oxide synthase activity was without effect. On the other hand, the capacity of more differentiated and less proliferative HT-29 cells for de novo L-arginine synthesis from L-citrulline was increased. It is concluded that L-citrulline is a precursor of L-arginine and L-ornithine in proliferative HT-29 cells and that the metabolic fate of L-ornithine in these cells is mainly devoted to polyamine synthesis. The similarity between differentiated HT-29 cells and the enterocytes of newborn animals in terms of L-arginine metabolism is finally discussed.

Differential inhibitory effects of three nitric oxide donors on ornithine decarboxylase activity in human colon carcinoma cells.

Ornithine decarboxylase (ODC, EC 4.1.1.17) is the enzyme responsible for the synthesis of polyamines, which are absolutely necessary for cell proliferation. In the present work, we tested the effects of 3 nitric oxide (NO) donors, namely, sodium nitroprusside (SNP), (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino] diazen-1-ium-1,2-diolate (MAHMA/NO) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO), on ODC activity in human-colon carcinoma cells (HT-29). SNP was the most effective inhibitor of ODC activity with a concentration of 8 micromol/L inducing 50% inhibition of basal activity. The effect of SNP was reversed by haemoglobin (Hb), but not by GSH or L-cysteine (CYS). Very little of the SNP in solution was degraded into nitrite, but the presence of cellular homogenate increased the production of nitrite. MAHMA/NO and DEA/NO were much less effective than SNP as ODC inhibitors, since the concentrations of these agents which induce 50% inhibition of basal activity were 20- to 60-fold higher than that of SNP. The effects of MAHMA/NO and DEA/NO were not reversed by haemoglobin. In solution, these latter 2 agents were totally degraded into nitrites. In conclusion, SNP on the one hand and MAHMA/NO and DEA/NO on the other appeared to release different NOx species with different efficiency on ODC activity.

Alpha-difluoromethylornithine (DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells.

Alpha-difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc-/+ cells, L-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to L-ornithine generation and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51+/-11% by 10 mM of DFMO whereas 10 mM of L-valine, a known potent arginase inhibitor, inhibited this flux by 73+/-6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated Ki (inhibitory constant) equal to 3.9+/-1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of L-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.

Effects of L-valine on growth and polyamine metabolism in human colon carcinoma cells.

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.

Effect of an elemental vs a complex diet on L-citrulline production from L-arginine in rat isolated enterocytes.

BACKGROUND: L-Arginine and L-glutamine are highly metabolized by intestinal cells, leading to various metabolites, including L-citrulline, which is required for optimal growth. Elemental diets, used in clinical practice to treat growth failure and malnutrition, are very different from complex diets normally consumed. The aim of the present study was to assess the effects of an elemental diet compared with a complex diet on L-arginine metabolism in rat isolated enterocytes and its modulation by L-glutamine. METHODS: Rats were fed the elemental diet (group ED) or the control diet (group C) for 14 days. Villus enterocytes then were isolated, and metabolic capacities or enzyme activities were assessed. RESULTS: The incubation of enterocytes isolated from group C with 0.1 mmol/L L-[U-14C]-arginine led to the production of 125 +/- 25 pmol L-citrulline/10(6) cells per 30 minutes. This production showed a twofold increase in the presence of 2 mmol/L L-glutamine. In group ED, L-citrulline synthesis from L-arginine was markedly lower in the absence or in the presence of L-glutamine. This coincided with lower carbamoylphosphate synthase I activity and carbamoylphosphate (CP) content of enterocytes. Other L-arginine and L-glutamine metabolic pathways were not affected. Similar results were obtained when the elemental diet was administered continuously through a gastric catheter or fed by mouth. CONCLUSIONS: L-Glutamine favors the synthesis of L-citrulline from L-arginine in isolated enterocytes, probably via an increase in CP production. Changing the diet composition, from a complex to an elemental diet, results in an alteration of the enterocyte capacity to synthesize L-citrulline from L-arginine, irrespective of the rhythm of delivery.

Histidine and histamine metabolism in rat enterocytes.

To study the metabolic fate of L-histidine and histamine in rat isolated enterocytes, enterocytes were incubated in the presence of 0.1 mM L-[U-14C] histidine. At the rate of 11.1 +/- 2.7 pmol/10(6) cells/30 min, the amino acid was incorporated into cellular proteins. 80 microM cycloheximide, i.e. a protein synthesis inhibitor, inhibited this incorporation by 70 +/- 17%. L-histidine was used for cellular protein synthesis which depended on time and concentration. 0.1 mM L-[U-14C] histidine was little oxidized by intestinal cells, i.e. 0.12 +/- 0.06 pmol/10(6) cells/30 min, and was not converted into histamine. When 10 mM histamine was added to the incubation medium, it completely inhibited the incorporation of 0.1 mM [1,4-14C] putrescine into isolated enterocytes. In enterocyte homogenates, this corresponded to inhibition by histamine of putrescine incorporation as catalyzed by transglutaminase activity. Since histamine incorporation into TCA-precipitable material derived from enterocyte homogenates depended on time and concentration, we concluded that exogenous, but not de novo-formed histamine was able to compete with putrescine incorporation into enterocytes as catalyzed by transglutaminase activity.