Effect of exogenous ATP upon inositol phosphate production, cationic fluxes and insulin release in pancreatic islet cells

Endogenous ATP is thought to play a key regulatory role in nutrient-stimulated insulin release. The present study deals with the effect of exogenous ATP and its stable analog alpha, beta-methylene ATP upon pancreatic islet function. Both alpha, beta-methylene ATP (5.0 microM to 0.2 mM) and ATP (0.3-3.0 mM) caused a rapid and concentration-related increase in insulin output by rat islets incubated or perfused at an intermediate concentration of D-glucose (8.3 mM). The effect of the ATP analog faded out at both lower and higher D-glucose concentrations. In the presence of 8.3 mM D-glucose, ATP also increased both 86Rb and 45Ca outflow from prelabelled islets. The cationic response to ATP persisted in the absence of extracellular Ca2+ and, hence, was reminiscent of that evoked by cholinergic agents. Like carbamylcholine, ATP caused a dose-related increase in the production of [3H]inositol phosphates from prelabelled islets or tumoral islet cells (RINm5F line). The latter effect was duplicated by alpha, beta-methylene ATP and unaffected by atropine. It is speculated that ATP, liberated together with insulin at the exocytotic site, might participate in a positive feedback control of insulin release.     

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.     

Effects of the Sequence of Isocaloric Meals with Different Protein Contents on Plasma Biochemical Indexes in Pigs

Nutrient composition and pattern of food intake may play a significant role in weight gain. The aim of this study was to document the effects of a daily 3-meal pattern with isocaloric diets containing different dietary protein contents on growth performance and different plasma biochemical indexes including amino acid plasma concentration in castrated male pigs. Then, 21 DLY (Duroc×Landrace×Yorkshire) pigs aged 60 days were assigned randomly into 3 groups: a control group (crude protein, CP 18.1%), a group receiving high then basal and then low CP meals (High-Low group) and a group receiving low then basal and then high CP meal (Low-High group) for 40 days with pigs being feed-restricted. On day 40, after 12 h fasting, blood samples were obtained for analysis. The results showed that the insulin/glucagon ratio was lower in the High-Low group (P<0.05) when compared with the control group. Compared with the control group, the average daily gain of pigs from the High-Low group increased by 14.10% (P = 0.046). Compared with the control group, serum gamma-glutamyl transferase (GGT) decreased significantly (P<0.05) in both the High-Low and Low-High groups. Plasma concentrations of branched-chain amino acids (BCAA: valine, isoleucine and leucine) increased in the Low-High group (P<0.05) when compared with the control group; and plasma methionine and serine decreased in both the two experimental groups (P<0.05). Compared with the High-Low group, all the BCAA increased significantly (P<0.05) in the Low-High group. These findings suggest that the sequence and quantity of alimentary protein intake affect the insulin/glucagon ratio, as well as amino acid concentrations including BCAA, methionine and serine. It is proposed that meal pattern with pigs receiving high then basal and then low CP meals daily may help to improve the weight gain of pigs.     

Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle

Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of L-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.     

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3?mg?kg^?1 lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.     

Stimulus-secretion coupling of arginine-induced insulin release. Metabolism of L-arginine and L-ornithine in pancreatic islets

Exogenous L-arginine and L-ornithine rapidly accumulate in rat pancreatic islets. L-Arginine is converted to L-ornithine and urea. Endogenous or exogenous L-ornithine generates di- and polyamines, the putrescine turnover being faster than that of spermidine and spermine. However, the major pathway for L-ornithine metabolism consists of its transamination to L-glutamaldehyde and further conversion to L-glutamate. The amines and L-glutamate derived from exogenous L-ornithine are incorporated into islet proteins at the intervention of transglutaminase and cycloheximide-sensitive biosynthetic processes, respectively. These findings suggest the hypothesis that the insulinotropic action of L-arginine and L-ornithine could somehow be related to the metabolism of these cationic amino acids in islet cells.     

Histidine and histamine metabolism in rat enterocytes

We have shown that the Metabolic Control Analysis (MCA) can explain the threshold effect observed in the expression of mitochondrial diseases [8]. As a matter of fact, the effect of a specific inhibitor on the flux of O2 consumption mimics a defect in a step of oxidative phosphorylation. The observed threshold is correlated to the value of the control coefficient of the inhibited step.For this reason, we have studied the repartition of the control coefficients of different steps in oxidative phosphorylation on various tissues (liver, kidney, brain, skeletal muscle and heart). We discuss the results in terms of metabolic control theory and provide a possible explanation for the heterogeneous phenotype of those pathologies. We present the double threshold hypothesis of both a threshold in the energy demand of a tissue and in the energy supply by oxidative phosphorylation. (Mol Cell Biochem 174: 143–148, 1997)     

Both Dietary Supplementation with Monosodium L-Glutamate and Fat Modify Circulating and Tissue Amino Acid Pools in Growing Pigs,but with Little Interactive Effect

Background

The Chinese population has undergone rapid transition to a high-fat diet. Furthermore, monosodium L-glutamate (MSG) is widely used as a daily food additive in China. Little information is available on the effects of oral MSG and dietary fat supplementation on the amino acid balance in tissues. The present study aimed to determine the effects of both dietary fat and MSG on amino acid metabolism in growing pigs, and to assess any possible interactions between these two nutrients.

Methods and Results

Four iso-nitrogenous and iso-caloric diets (basal diet, high fat diet, basal diet with 3% MSG and high fat diet with 3% MSG) were provided to growing pigs. The dietary supplementation with fat and MSG used alone and in combination were found to modify circulating and tissue amino acid pools in growing pigs. Both dietary fat and MSG modified the expression of gene related to amino acid transport in jejunum.

Conclusions

Both dietary fat and MSG clearly influenced amino acid content in tissues but in different ways. Both dietary fat and MSG enhance the absorption of amino acids in jejunum. However, there was little interaction between the effects of dietary fat and MSG.