Pierre-Henri Duée

Drastic changes in fecal and mucosa-associated microbiota in adult patients with short bowel syndrome

Short bowel syndrome (SBS) is observed in Humans after a large resection of gut. Since the remnant colon and its associated microbiota play a major role in the outcome of patients with SBS, we studied the overall qualitative and quantitative microbiota composition of SBS adult patients compared to controls. The population was composed of 11 SBS type II patients (with a jejuno-colonic anastomosis) and 8 controls without intestinal pathology. SBS patients had 38 +/- 30 cm remnant small bowel length and 66 +/- 19% of residual colon. The repartition of proteins, lipids, carbohydrates and fibres was expressed as % of total oral intake in patients and controls. The microbiota was profiled from stool and biopsy samples with temporal temperature gradient gel electrophoresis and quantitative PCR. We show here that microbiota of SBS patients is unbalanced with a high prevalence of Lactobacillus along with a sub-dominant presence and poor diversity of Clostridium leptum, Clostridium coccoides and Bacteroidetes. In addition, Lactobacillus mucosae was detected within the fecal and mucosa-associated microbiota of SBS patients, whereas it remained undetectable in controls. Thus, in SBS the microbial composition was deeply altered in fecal and mucosal samples, with a shift between dominant and sub-dominant microbial groups and the prevalence of L. mucosae.

Arginine metabolism in rat enterocytes

Rat enterocytes exposed to L-arginine in the absence of any other exogenous substrate were found to actively metabolize this cationic amino acid. L-Arginine was converted to L-citrulline either directly in a NADPH-sensitive manner thought to be coupled with the generation of NO, or indirectly through the sequence of reactions catalyzed by arginase and ornithine transcarbamylase. A large fraction of L-citrulline and L-ornithine generated from exogenous L-arginine was released in the incubation medium. The production of CO2 and (poly)amines from L-arginine occurred at rates 2 to 3 orders of magnitude lower than that characterizing the net uptake of the cationic amino acid, and this despite the fact that enterocytes were equipped to allow the interconversion of L-ornithine and L-glutamate. It is concluded that the oxidative catabolism of L-arginine in enterocytes is quantitatively negligible relative to its conversion to L-citrulline and L-ornithine.

Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome

In short bowel syndrome (SBS), although a remaining colon improves patient outcome, there is no direct evidence of a mucosal colonic adaptation in humans. This prospective study evaluates morphology, proliferation status, and transporter expression level in the epithelium of the remaining colon of adult patients compared with controls. The targeted transporters were Na+/H+ exchangers (NHE2 and 3) and oligopeptide transporter (PepT1). Twelve adult patients with a jejuno-colonic anastomosis were studied at least 2 yr after the last surgery and compared with 11 healthy controls. The depth of crypts and number of epithelial cells per crypt were quantified. The proliferating and apoptotic cell contents were evaluated by revealing Ki67, PCNA, and caspase-3. NHE2, NHE3, PepT1 mRNAs, and PepT1 protein were quantified by quantitative RT-PCR and Western blot, respectively. In patients with SBS compared with controls, 1) hyperphagia and severe malabsorption were documented, 2) crypt depth and number of cells per crypt were 35% and 22% higher, respectively (P < 0.005), whereas the proliferation and apoptotic levels per crypt were unchanged, and 3) NHE2 mRNA was unmodified; NHE3 mRNA was downregulated near the anastomosis and unmodified distally, and PepT1 mRNA and protein were unmodified. We concluded that, in hyperphagic patients with SBS with severe malabsorption, adaptive colonic changes include an increased absorptive surface with an unchanged proliferative/apoptotic ratio and well-preserved absorptive NHE2, NHE3, and PepT1 transporters. This is the first study showing a controlled nonpharmacological hyperplasia in the colon of patients with SBS.

Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-Butyrate, glucose, and glutamine

BACKGROUND & AIMS Among substrates available to the colonic mucosa, n-butyrate from bacterial origin represents a major fuel. The present work investigated possible modifications of energy substrate metabolism in colonocytes isolated from germfree rats. METHODS Colonocytes isolated from germfree vs. conventional rats were incubated (30 minutes at 37 degrees C) in the presence of 14C-labeled n-butyrate (10 mmol/L), glucose (5 mmol/L), or glutamine (5 mmol/L). 14CO2 and metabolites generated were measured. Possible regulatory steps were also investigated. RESULTS Glucose use rate was 25% lower in germfree rat colonocytes due to a reduced glycolytic capacity in these cells. Differences in 6-phosphofructo-1-kinase activity could account for this decrease. In contrast, glutamine use rate was 45% higher, and this was correlated with a higher maximum velocity of glutaminase in these cells. Nevertheless, the capacities to oxidize glucose and glutamine remained unchanged. Although the capacity to use n-butyrate was maintained in colonocytes of germfree rats, the ketogenic capacity was lower, whereas the capacity to oxidize n-butyrate was higher. The mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein was identified in the colonic mucosa. Moreover, the messenger RNA and amount of protein were 75% lower in the germfree state. CONCLUSIONS The absence of an intestinal microflora induces specific changes in the metabolic capacities of colonocytes.

Polyamine metabolism in enterocytes isolated from newborn pigs

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.

Fuel selection in intestinal cells

Compte tenu de ses fonctions multiples, l’intestin, premiere cible de l’aliment, joue un r61e crucial dans le maintien de 1’Ctat de sante. Les fonctions d’hydrolyse et de transport assurees par les cellules intestinales differenciees, et d’une faGon gCnCrale, 1’intCgritC des membranes intestinales nkcessitent un metabolisme intense de certains nutriments, d’origine luminale et/ou vasculaire. La demande CnergCtique est, en effet, tres Clevee puisque la consommation intestinale d’O2 s’Clkve B 20-25% de la consommation totale de l’organisme. Parmi les nutriments, qui rendent compte de cette consommation energetique ClevCe, le r6le de la glutamine a ete demontre. Les raisons pour lesquelles cet acide amink, prelevC du c6tC luminal (avec le glutamate et l’aspartate) comme du c6te vasculaire est utilisC d’une fagon privilkgiee, sont CvoquCes (activite et affinitC de la glutaminase (EC 3.5.1.2), decarboxylation incomplete de la glutamine conduisant B la generation de composes a trois carbones, interactions metaboliques avec les autres nutriments). Par ailleurs, Ie faible r81e Cnergetique dans l’intestin grele du glucose et des acides gras B longue chaine est expliquk. NCanmoins, dans la partie distale de l’intestin (colon), les acides gras a courte chaine, et plus particulierement, le butyrate, representent des substrats CnergCtiques majeurs des cellules Cpithiliales. Enfin, les modifications du mCtabolisme CnergCtique des cellules intestinales en fonction de l’iige et de l’Ctat pathologique sont abordCes. L’adaptation du metabolisme CnergCtique de l’intestin grsle au cours du developpement fait ainsi apparaitre des voies metaboliques transitoires telles que la voie de la gluconiogenkse et de la cktogenese.

Intestinal Oxygen Uptake and Glucose Metabolism During Nutrient Absorption in the Pig

Abstract Intestinal transport of nutrients coincides with their partial catabolism in the gut. The aim of the present study was to measure intestinal oxygen consumption and nutrient metabolism after a meal or during a short fast. Nutrient and oxygen balances across the small intestine were measured in consclous 50 kg (live wt) pigs. Jejunal enterocytes were also isolated from 1-hr postprandial, postabsorptive, or 3-day fasted pigs, in order to evaluate their capacities to metabolize 5 mM glucose and 2 mM glutamine. Whatever the nutritional state, intestinal oxygen consumption was high, since 26 ± 2% (n = 6) of the oxygen arterial supply was extracted by the small intestine. Furthermore, the consumption of a mixed meal induced a rapid and transient rise in oxygen consumption. In the postabsorptive state, the intestinal uptake of glucose (0.31 ± 0.08 mmole/min, n = 6) was twice higher than that of glutamine. The role of glucose as a fuel was also evidenced after a 3-day fast. During nutrient absorption, glutamine was highly utilized, and lactate was produced. The capacity of enterocytes isolated from fed pigs to metabolize glucose was dramatically reduced, as was 6-phosphofructo 1-kinase activity. In contrast, intestinal muscle presented a high glycolytic capacity from glucose, suggesting that the main site of intestinal lactate production during nutrient absorption would be the muscular rather than the epithelial layer.

Metabolism of l-arginine through polyamine and nitric oxide synthase pathways in proliferative or differentiated human colon carcinoma cells

HT-29 Glc-/+ cells originate from a human colon adenocarcinoma. These cells have been selected in a glucose-free culture medium and switched back in a glucose-containing medium. In this condition, they can spontaneously differentiate after confluency in enterocyte-like cells according to the activity of the brush-border associated hydrolase dipeptidyl peptidase IV. Since L-arginine can generate polyamines which are necessary for cellular proliferation and also differentiation, and nitric oxide with reported anti-proliferative property, the metabolism of this amino acid was examined in proliferative and differentiated isolated HT-29 cells. Proliferative HT-29 cells were characterized by micromolar intracellular concentration of putrescine and millimolar concentration of spermidine and spermine. In these cells, L-arginine is converted to L-ornithine and putrescine and to a minor part to nitric oxide and L-citrulline. Putrescine was taken up by HT-29 cells, leading to the production of a modest amount of spermidine. The diamine was slightly incorporated into cellular proteins and largely released in the incubation medium. The proliferative HT-29 cells take up spermidine and spermine but do not catabolize these polyamines and slightly released spermidine. Differentiation of HT-29 cells is not associated with change in intracellular polyamine content but is paralleled by an almost complete extinction of de novo synthesis of putrescine (due to a dramatic decrease of ornithine decarboxylase activity) and by a reduced release capacity of putrescine. In contrast, putrescine net uptake and incorporation into cellular proteins remained unchanged after differentiation. Furthermore, spermidine and spermine metabolism as well as the circulation of L-arginine in the nitric oxide synthase pathway were also not modified after differentiation. In conclusion, putrescine is the L-arginine-derived molecule, the metabolism of which is specifically and markedly modified when HT-29 cells move from proliferative to differentiated state.

Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat

Previous studies have suggested that intestinal microbiota modulates colonic epithelium renewal. The objective of our work was to study the effects of microbiota on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic crypts and amount of cell cycle-related proteins were compared between germ-free (GF), conventional (CV), and conventionalized rats by histochemistry and Western blot. Ki67 and proliferating cell nuclear antigen (PCNA) were used as surrogates for proliferative cells; p21(cip1) and p27(kip1) were markers of cell cycle arrest; anti- and proapoptotic proteins, Bcl2 and Bax, respectively, were also studied. We observed 40% increase of the crypt proliferative area 2 days after inoculation of GF rats with a complex microbiota. This recruitment of proliferative cells may account for the 30% increase of crypt depth observed between CV and GF rats. The hyperproliferative boost induced by microbiota was compensated by a fourfold increase of p21(cip1) and p27(kip1) involved in cell cycle arrest and a 30% drop of antiapoptotic Bcl2 protein while Bax was unchanged. Inductions of p21(cip1), p27(kip1), and PCNA protein were not paralleled by an increase of the corresponding mRNA. We also showed that p21(cip1) induction by microbiota was partially restored by Bacteroides thetaiotaomicron, Ruminococcus gnavus, and Clostridium paraputrificum. Colonization of the colon by a complex microbiota increases the crypt depth of colon epithelium. This event takes place in conjunction with a multistep process: a hyperproliferative boost accompanied by compensatory events as induction of p21(cip1) and p27(kip1) and decrease of Bcl2.

Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells

In human colon carcinoma HT‐29 Glc−/+ cells, l‐arginine is the common precursor of polyamines which are absolutely necessary for cellular proliferation and nitric oxide (NO) with reported anti‐proliferative activity. The aim of the present work was to test the effect of the NO donor sodium nitroprusside (SNP) on polyamine synthesis and cellular growth in HT‐29 cells. SNP in the micromolar range inhibits cellular putrescine synthesis and this effect is greatly reversed by haemoglobin, supporting the view that the effect of SNP is related to the generation of NO. This corresponds to the inhibition by SNP of ornithine decarboxylase activity. Furthermore, SNP inhibits cellular proliferation. The effect of SNP is reversed by haemoglobin after 2 days of treatment but not after 4 days. Although no acute toxic effect of SNP was detected after 90 min incubation, it greatly enhanced the cellular death rate after several days in culture as estimated by the LDH leakage test. In conclusion, our data raise the possibility of an inhibitory interrelationship between NO and polyamine metabolic pathways. NO induced inhibition of putrescine synthesis and growth in HT‐29 cells is discussed from a causal perspective.