Marie-Thérèse Morel

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.

Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by NH4+

Short chain fatty acids (SCFA) from bacterial origin, as well as glucose from vascular origin, are among fuel substrates available to the colonic mucosa. The present work investigated the possible modulation by another bacterial metabolite, i.e. ammonia, of the capacities of colonic epithelial cells to metabolize these substrates. Viable colonocytes were isolated from the proximal colon of 40–50 kg pigs fed a standard diet and were incubated (30 min, 37°C) in the presence of a concentration range of 14C-labeled n-butyrate or acetate, or 14C-labeled glucose (5 mm), with or without NH4Cl (10 mM) addition. 14CO2 and metabolites generated were measured. Butyrate utilization resulted in a high generation of ketone bodies (acetoacetate and β-OH-butyrate), in addition to 14CO2 production. However, the net ketone body generation was significantly decreased for butyrate concentrations higher than 10 mM. In contrast to n-butyrate, acetate when given as the sole substrate got preferentially metabolized in the oxidation pathway. Acetate metabolism was not affected by NH4Cl, thus indicating that the tricarboxylic acid cycle was unchanged. Conversely, 4C02 and ketone body production from butyrate were decreased by 30% in the presence of NH4Cl, suggesting that butyrate activation or β-oxidation was diminished. Glucose utilization rate was increased by 20%, due to an increased glycolytic capacity in the presence of NH4Cl. A dose-dependent stimulation of phosphofructokinase activity by NH4+ could account for this effect. It is concluded that ammonia, whose physiological concentration is high in the colonic lumen, can modulate the metabolism of two major substrates, n-butyrate and glucose, in colonic epithelial cells.

Glucose, galactose, and glutamine metabolism in pig isolated enterocytes during development

ABSTRACT: In the pig, the gastrointestinal tract grows rapidly after birth and undergoes a short postnatal maturation. The objective of the present work was to assess the metabolic characteristics of the small intestinal mucosa during this period by investigating glucose, galactose, and glutamine metabolism in pig isolated enterocytes. Piglets were used immediately after birth or at various stages during suckling or postweaning. Fed animals were taken in a postabsorptive state. The jejunoileum was excised and perfused with an EDTA (5 mM)-containing buffer. The epithelial cell layer was further dissociated in the presence of hyaluronidase (0.01%). The resulting cell suspension (95% absorbing enterocytes; viability greater than 90%) was incubated with 14C-labeled substrates to measure 14CO2 production in parallel with substrate disappearance. The capacity to utilize glutamine was high and remained steady during the suckling period. Glucose utilization capacity was limited at birth and increased more than 3-fold during the first week of suckling. Such an increase was not observed in piglets kept unsuckled since birth. Galactose utilization capacity remained steady during the first week but afterward gradually disappeared. Lactate and pyruvate production through glycolysis was the major pathway accounting for glucose or galactose disappearance. A capacity for a net glucose production from galactose was evidenced during the first week of suckling. Thus, isolated newborn pig enterocytes exhibit specific and transient metabolic characteristics during the first postnatal week.

Characterization and ontogenesis of nitric oxide synthase activity in pig enterocytes

Nitric oxide has been implicated as a local modulator of several gastrointestinal functions. In this study, we have measured nitric oxide synthase activity in homogenates of enterocytes isolated from post‐weaned pigs. The enzyme required the presence of NADPH and 6‐(R,S)‐5,6,7,8‐tetrahydro‐l‐biopterin. Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca2+ or EGTA and was inhibited by the arginine analogs N G‐monomethyl‐l‐arginine and N ω‐nitro‐l‐arginine. NO synthase activity was not detectable in enterocytes isolated at birth and increased slightly in suckling animals. NO synthase activity was found to be present mostly in the cytosolic fraction isolated from post‐weaned pigs enterocytes.

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.

Control of glucose metabolism in newborn pig enterocytes: Evidence for the role of hexokinase

The objective of the present work was to identify the regulatory step(s) in the post-natal development of a high glycolytic capacity previously evidenced in newborn pig enterocytes (Darcy-Vrillon et al. (1994) Pediat. Res., 36, 175-181. Glucose entry via the Na+/glucose cotransporter, estimated by the uptake of the non-metabolizable analogue methyl alpha-D-[U-14C]glucopyranoside, slightly decreased between birth and 2 days of sucking. The flux of glucose metabolized into the pentose cycle pathway slightly increased but could not account for the 3-fold increase observed in the glycolytic capacity. Whereas the maximal activity of 6-phosphofructo-1-kinase did not change between stages, there was a significant increase in hexokinase activity as well as in the flux of glucose phosphorylated. These findings suggest that the stimulation of glucose phosphorylation through hexokinase is the key event leading to an increased glycolytic capacity of small intestinal cells at the onset of sucking.

Effect of an Elemental vs a Complex Diet on Polyamine Metabolism in Rat Isolated Enterocytes

BACKGROUND Polyamines play an important role in the proliferation and differentiation of enterocytes. Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis. Elemental diets, providing easily absorbable nutrients such as free amino acids, are used in clinical practice to treat growth failure and malnutrition. They are very different from complex diets normally consumed. Little information is available about the influence of elemental diets on metabolic capacities of enterocytes. This study was undertaken in rats to assess the effects on polyamine metabolism of an elemental diet compared with a complex diet. METHODS Rats were fed the elemental diet (group ED) or the control diet (group C) for 14 days. The dietary intakes were isocaloric and isonitrogenous in groups C and ED. Villous enterocytes were then isolated and metabolic capacities or enzyme activities were assessed. RESULTS Both the enterocyte capacity to decarboxylate ornithine through ODC (measured in viable enterocytes) and ODC activity (measured in homogenates) were severely decreased in group ED. The polyamine content in enterocytes, however, was maintained at a similar level in both groups. This coincided with a decrease in the main enzymatic activity responsible for putrescine catabolism (ie, diamine oxidase activity) in group ED. CONCLUSIONS Although nutrition manipulation was shown to alter polyamine biosynthesis in this study, the polyamine homeostasis was probably maintained, at least in part, through down-regulation of diamine oxidase.