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.

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.

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.

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.

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.

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.

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 NG-monomethyl-L-arginine and N omega-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.

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.

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.

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.

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.

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.

Nutritional and clinical aspects of parenteral nutrition in pigs irradiated on the abdomen with supralethal doses.

Nutritional balances of nitrogen and minerals (sodium potassium, calcium, phosphorus) were established in 10 pigs of 40 kg receiving a continuous parenteral nutrition supplying a daily amount of 1500 ml containing 1500 kcal and 2.2-5.4 g of protein, and in addition, 2 liters of water per day. The balances were recorded for 5 days in the animals used as controls and for 9 days in those irridated with 1000 rd in the median plane. This dose represents the inferior limit for appearence of the gastrointestinal syndrome in this species. The irradiated subjects seemed to be able to use a relatively high supply of energy and protein. Water and nitrogen balances were easy to obtain with respect to sodium, potassium and calcium, whereas the deficiency in phosphorus was difficult to compensate for through the intravenous route only.

Contribution of hepatic fatty acid oxidation and exogenous galactose supply to the regulation of glucose homeostasis in the newborn rabbit.

The inhibition of hepatic gluconeogenesis by 3-mercaptopicolinic acid (3-MPA) leads to a profound hypoglycemia in both suckling and fasting 24-hour-old rabbits. This hypoglycemia is totally reversed 1 h after the intragastric injection of an amount of galactose corresponding to the one ingested daily by the suckling newborns. This results from an active gluconeogenesis from galactose, which bypasses the site of inhibition by 3-MPA. However, this amount of galactose is not sufficient to maintain a normal blood glucose concentration for a long time, since 3 h after galactose injection, the blood glucose concentrations of newborn rabbits return to hypoglycemic values. When hepatic fatty acid oxidation is inhibited by 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (POCA), 24-hour-old fasting rabbits become rapidly hypoglycemic secondary to a decrease in liver gluconeogenesis. The rate of hepatic gluconeogenesis is totally restored by giving medium-chain triglycerides, and the 24-hour-old rabbits become normoglycemic.

Influence of environmental temperature on glucose tolerance and insulin response in the new-born piglet.

Plasma glucose and insulin concentrations were measured in 45 new-born piglets both before and at regular intervals throughout a 2-hour period following an intravenous administration of glucose (1 g/kg body weight), at environmental temperatures of 17, 24 and 33 degrees C. As anticipated, rectal temperature (TR was dependent upon the environmental temperature. During the glucose tolerance test (GTT), TR remained constant at both 24 and 33 degrees C, 37.0 +/- 0.2 compared with 38.0 +/- 0.1 degrees C. However at 17 degrees C, 12 piglets were able to maintain a constant TR (35.8 +/- 0.2 degrees C), whereas the remaining 6 piglets became hypothermic (32.8 +/- 0.4 degrees C), with TR decreasing continuously throughout the GTT. Their body weight was 210 g below that of the other piglets (p less than 0.05). Those piglets at 17 and 24 degrees C which maintained homeothermy had a higher glucose disappearance rate (KG; 2.00 and 2.31%/min, respectively) than those kept within thermal neutrality (1.66%/min). The insulin response to glucose was similar. During hypothermia, on the other hand, both KG (0.76 +/- 0.12%/min) and the insulin response were significantly less (p less than 0.05) than in all other piglets. These results indicate that the glucose uptake by the skeletal muscle is increased in cold-exposed, normothermic piglets and results in an increased metabolic rate. Failure to maintain homeothermy diminishes glucose uptake and inhibits insulin release.

Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes.

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

Stimulation by D-glucose of the direct conversion of arginine to citrulline in enterocytes isolated from pig jejunum.

In enterocytes isolated from pig jejunum, L-arginine is metabolized to L-citrulline either directly or indirectly through the sequence of reactions catalysed by arginase and ornithine transcarbamylase. In the presence of 5 mM D-glucose, the direct conversion of 1mM L-[guanido-14C] arginine to L-citrulline was increased more than 4 times. Isolated enterocytes exhibit a high glycolytic capacity. Furthermore, the decarboxylation of 5mM D-[1-14C] glucose was 3.6 fold higher than the decarboxylation of 5 mM D-[6-14C] glucose which suggests the presence of a pentose phosphate pathway in enterocytes. Since the production of labelled L-citrulline from L-[guanido-14C] arginine in pig enterocyte homogenates was markedly increased in the presence of NADPH, it is proposed that the direct conversion of L-arginine to L-citrulline could be stimulated by the production of NADPH from D-glucose in the pentose phosphate pathway.