Colonic delivery of sodium butyrate via oral route: acrylic coating design of pellets and in vivo evaluation in rats.

Few pharmaceutical studies, with the exception of those on rectal solutions, are described on short chain fatty acid (SCFA) formulations-especially for sodium butyrate, which is a colonocyte preferential substrate. Highly dosed butyrate pellets (90%) were prepared and their coating was designed for colonic delivery. In vivo determination (pH and transit time of pellets in rats) allowed to respectively choose the grade and thickness (resistance of 6 h) of the pH-dependent coating (Eudragi L+S, 1:1). The coated pellets were administered to naturally butyrate-deprived rats. The rats' colonic mucosa had the particularity to weakly express mitochondrial HMG-CoA synthase, an enzyme that responds to luminal butyrate. The results did not show early absorption of butyrate, but a probable cecal loss in the rat cecum as cecal residence time of the pellets was important and as pH was propitious for the coating hydrolysis. It seemed that butyrate, given daily for 7 days without the other main SCFA. was unable to induce the enzyme and/or that the dose (0.32 mmol/day) was insufficient.

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.

Non-starch polysaccharides extracted from seaweed can modulate intestinal absorption of glucose and insulin response in the pig.

We have investigated the possible effects of algal polysaccharides on postprandial blood glucose and insulin responses in an animal model, the pig. Three seaweed fibres of different viscosities, extracted from Palmaria palmata (PP), Eucheuma cottonii (EC), or Laminaria digitata (LD), were compared to purified cellulose (CEL). Blood glucose and plasma insulin levels were monitored and intestinal absorption quantified for 8 h following a high carbohydrate test-meal supplemented with 5% fibre. Digestive contents were also sampled, 5 h postprandial. As compared to CEL, PP had no effect on glucose and insulin responses. The latter decreased with EC, but glucose absorption balance was not modified. LD addition resulted in a dramatically reduced glucose absorption balance, accompanied by a higher amount of starch left in the small intestine. Among polysaccharides tested, only the highly viscous alginates could affect intestinal absorption of glucose and insulin response.

Dietary glutamine suppresses endogenous glutamine turnover in the rat.

Plasma glutamine turnover was determined using 1-14C-labeled glutamine in rats that consumed crystalline amino acid diets containing the equivalent of 16% protein with 25% of the amino acids as glutamine or a control diet containing no glutamine (or glutamate) for 10 days. Glutamine turnover in glutamine-fed animals was 66% of the rate in the control group. Glutamine feeding caused 20% higher levels of arterial plasma glutamine. Arterial-portal differences across the portal-drained viscera showed net glutamine uptake in control animals but no net uptake or release in the glutamine-fed group. Skeletal muscle glutamine synthetase activity was similar in both groups. The results indicate that long-term consumption of relatively large amounts of dietary glutamine decreases the turnover of plasma glutamine and thus reduces the need for endogenous glutamine synthesis.

Euglycemic hyperinsulinemic clamp to assess posthepatic glucose appearance after carbohydrate loading. 1. Validation in pigs.

BACKGROUND: Precise knowledge of the rate of glucose absorption after meal feeding requires invasive methods in humans. OBJECTIVE: This study aimed to validate in an animal model a technique combining the euglycemic hyperinsulinemic clamp and oral carbohydrate loading (OC-Clamp) as a noninvasive procedure to quantify the posthepatic appearance of glucose after oral carbohydrate loading. DESIGN: Twenty-one pigs were fitted with arterial, jugular, portal, and duodenal catheters and a portal blood flow probe. At glucose clamp steady state, duodenal glucose (0.9 g/kg; DG-Clamp) and oral carbohydrate (140 g corn or mung bean starch as part of a mixed meal; OC-Clamp) were administered while the glucose infusion was progressively reduced to compensate for the incremental posthepatic appearance of glucose. [3-3H]glucose was used to assess the glucose turnover rate. RESULTS: Hepatic glucose production was totally suppressed by insulin infusion, and the whole-body glucose turnover rate remained stable during glucose absorption. The incremental portal appearance of glucose after the DG load was not altered by hyperinsulinemia, and the cumulative posthepatic appearance of glucose was 63 +/- 3% (x +/- SEM) of the DG load. The net hepatic portal appearance of glucose remained constant during absorption (34 +/- 3% of the load). After the OC load, the respective portal appearance rates of glucose were significantly different between carbohydrate sources; however, the rates paralleled those of the posthepatic appearance of glucose. Again, net hepatic glucose uptake expressed as portal appearance was similar for both carbohydrates. CONCLUSIONS: The results validate the OC-Clamp method to monitor the posthepatic appearance of glucose after carbohydrate ingestion and to discriminate between different carbohydrate sources. The results suggest that the technique be used in humans.

Euglycemic hyperinsulinemic clamp to assess posthepatic glucose appearance after carbohydrate loading. 2. Evaluation of corn and mung bean starches in healthy men.

BACKGROUND: The rate of absorption of glucose from carbohydrates is important in several aspects of health. We recently validated a noninvasive technique in pigs, euglycemic hyperinsulinemic clamp plus oral carbohydrate loading (OC-Clamp), to quantify the rate of net posthepatic appearance of glucose after ingestion of carbohydrates. OBJECTIVE: The OC-Clamp procedure was performed in 8 healthy men to compare the net posthepatic appearance of glucose after ingestion of 1 of 3 carbohydrates. DESIGN: Human volunteers underwent the OC-Clamp procedure at an insulin infusion rate of 1.5 mU x kg(-1) x min(-1) (n = 5). The oral carbohydrate load (1 g/kg) consisted of glucose, cornstarch, or mung bean starch. During the OC-Clamp procedure, the glucose infusion rate decreased during absorption to maintain plasma glucose steady state and the decrease reflected the net posthepatic appearance of glucose. In addition, carbohydrates were loaded without insulin infusion (n = 6) and glycemic indexes were calculated (with glucose as the reference). RESULTS: The mean (+/-SEM) glycemic index of cornstarch was higher (95 +/- 18) than that of mung bean starch (51 +/- 13). In the OC-Clamp experiments, the posthepatic appearance of glucose and cornstarch did not differ significantly and represented 79.4 +/- 5.0% and 72.6 +/- 4.0%, respectively, of the load after complete absorption (within 3 h). In contrast, the net posthepatic appearance of glucose from mung bean starch was significantly lower (35.6 +/- 4.6% of the load, P < 0.001) than that from glucose and cornstarch, even 4.5 h postprandially. CONCLUSIONS: The OC-Clamp technique allows a continuous assessment of net posthepatic appearance of glucose after ingestion of carbohydrates and significant discrimination between corn and mung bean starches.

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.

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.

GLUT2 and hexokinase control proximodistal gradient of intestinal glucose metabolism in the newborn pig.

We have reported previously that a high glycolytic capacity develops soon after birth in enterocytes isolated from suckling newborn pigs. In the present work, we investigated whether such metabolic changes could affect intestinal glucose utilization in vivo and examined possible variations in glucose metabolism along the small intestine. Glucose utilization by individual tissues was assessed using the 2-deoxyglucose technique. The overall glucose utilization rate was doubled in suckling vs. fasting 2-day-old pigs because of significantly higher rates in all tissues studied, except for the brain. In parallel, enterocytes were isolated from the proximal, medium, or distal jejunoileum of newborn vs. 2-day-old pigs and assessed for their capacity to utilize, transport, and phosphorylate glucose. Intestinal glucose consumption accounted for approximately 15% of glucose turnover rate in suckling vs. 8% in fasting pigs. Moreover, there was a proximal-to-distal gradient of glucose utilization in the intestinal mucosa of suckling pigs. Such a gradient was also evidenced on isolated enterocytes. The stimulation of both hexokinase activity (HK2 isoform) and basolateral glucose transporter (GLUT2), as observed in the proximal jejunum, could account for such a site-specific effect of suckling.

Parenteral nutrition modifies glucose and glutamine metabolism in rat isolated enterocytes.

BACKGROUND & AIMS: After small bowel resection, parenteral nutrition is often required to provide energy and nitrogen supplies and also to stimulate intestinal adaptation, despite the absence of glutamine in formulas. The aim of this study was to investigate the effect of nutrient supply route on fuel utilization by enterocytes. METHODS: Rats received an intravenous or intragastric continuous infusion of an all-in-one glutamine-free formula. Sham-operated control rats were orally fed and received the same protein-caloric supplies as the other two groups. On day 7, the rats were killed in the fed state, blood samples were collected, and the jejunoileum was removed. Enterocytes were isolated. Aliquots of cell suspensions were incubated (30 minutes at 37 degrees C) in the presence of [14C]glucose and [14C]glutamine (2 mmol/L). Substrate utilization was determined by measuring metabolites and CO2 generated. RESULTS: Intravenously fed rats showed mild hyperglycemia and marked hyperinsulinemia. Plasma glutamine levels were similar in the three groups. Intravenously fed rats showed a simultaneous increase in glutamine utilization and a decrease in glucose utilization compared with intragastrically fed and control rats, without parallel changes in glutaminase and hexokinase activities. The basolateral glucose transporter protein concentration was reduced in intravenously fed rat enterocytes. CONCLUSIONS: The route of nutrient delivery influences fuel utilization by enterocytes.

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.

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 degrees 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 beta-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, 14CO2 and ketone body production from butyrate were decreased by 30% in the presence of NH4Cl, suggesting that butyrate activation or beta-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.

Evidence for increased anionic arginase activity in pig enterocytes during development.

We have recently reported that the flux of L-arginine through arginase in enterocytes is increased in weaned pigs when compared with suckling animals (Blachier et al. 1993, Eur. J. Biochem. 216, 109-117). The aim of the present study was to characterize arginase activities at both stages of development. Enterocytes isolated from suckling animals were found to possess an anionic (50%) and a non-anionic (50%) form of arginase as judged from activities recovered from DEAE-cellulose ion exchange chromatography. In enterocytes isolated from weaned animals, anionic arginase was the major form representing 89% of arginase activity. This isoform is characterized by increased affinity for L-arginine (2 fold) and increased maximal velocity (39 fold) when compared with the anionic form originating from suckling piglet enterocytes. In conclusion, our data demonstrate that pig enterocytes are equipped with at least 2 isoforms of arginase and that anionic form of arginase activity appeared to be mainly responsible for the capacity of weaned pig enterocytes to catabolize L-arginine.

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.

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.

Transglutaminase activity in enterocytes isolated from pig jejunum.

Polyamines appear to be involved in the turnover, growth and maintenance of intestinal mucosa integrity. Since polyamines could act -in part at least- through their incorporation into cellular proteins as catalyzed by transglutaminase, we have measured this enzyme activity in villus enterocytes isolated from pig jejunum and in homogenate derived from isolated cells. A part of putrescine, spermidine and spermine taken up by enterocytes is incorporated in TCA precipitable material derived from cells and this corresponds to the presence of transglutaminase activity in cellular homogenates. This activity which is time and substrate concentration dependent is strongly inhibited by the transglutaminase inhibitor glycine methyl ester. The capacity for de novo production of polyamines from L-arginine or L-glutamine is very limited in isolated enterocytes, and this coincided with a very low ornithine decarboxylase activity when compared with polyamine cell content. It is concluded that the main source of polyamines for pig enterocytes is extracellular and that exogenous polyamines are substrates for enterocyte transglutaminase.

Intestinal oxygen uptake and glucose metabolism during nutrient absorption in the pig.

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 conscious 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.