Differential changes to splanchnic and peripheral protein metabolism during the diet-induced development of metabolic syndrome in rats.

Little is known about the effects of the development of metabolic syndrome (MS) on protein and amino acid (AA) metabolism. During this study, we took advantage of the variability in interindividual susceptibility to high fat diet-induced MS to study the relationships between MS, protein synthesis, and AA catabolism in multiple tissues in rats. After 4 mo of high-fat feeding, an MS score (ZMS) was calculated as the average of the z-scores for individual MS components [weight, adiposities, homeostasis model for the assessment of insulin resistance (HOMA-IR), and triglycerides]. In the small intestine, liver, plasma, kidneys, heart, and muscles, tissue protein synthesis was measured by 2H2O labeling, and we evaluated the proportion of tissue AA catabolism (relative to protein synthesis) and nutrient routing to nonindispensable AAs in tissue proteins using natural nitrogen and carbon isotopic distances between tissue proteins and nutrients (Δ15N and Δ13C), respectively. In the liver, protein mass and synthesis increased, whereas the proportion of AA catabolism decreased with ZMS. By contrast, in muscles, we found no association between ZMS and protein mass, protein synthesis (except for a weak positive association in the gastrocnemius muscle only), and proportion of AA catabolism. The development of MS was also associated with altered metabolic flexibility and fatty acid oxidation, as shown by less routing of dietary lipids to nonindispensable AA synthesis in liver and muscle. In conclusion, MS development is associated with a greater gain of both fat and protein masses, with higher protein anabolism that mainly occurs in the liver, whereas muscles probably develop anabolic resistance due to insulin resistance.

Diet-animal fractionation of nitrogen stable isotopes reflects the efficiency of nitrogen assimilation in ruminants.

The natural abundance of ¹5N in animal proteins (δ¹5Nanimal) is greater than that in the diet consumed by the animals (δ¹5Ndiet), with a discrimination factor (Δ¹5N = δ¹5Nanimal - δ¹5Ndiet) that is known to vary according to nutritional conditions. The objectives of the present study were to test the hypothesis that Δ¹5N variations depend on the efficiency of nitrogen utilisation (ENU) in growing beef cattle, and to identify some of the physiological mechanisms responsible for this N isotopic fractionation in ruminants. Thus, we performed the regression of the Δ¹5N of plasma proteins obtained from thirty-five finishing beef cattle fed standard and non-conventional diets against different feed efficiency indices, including ENU. We also performed the regression of the Δ¹5N of different ruminant N pools (plasma and milk proteins, urine and faeces) against different splanchnic N fluxes obtained from multi-catheterised lactating dairy cows. The Δ¹5N of plasma proteins was negatively correlated with feed efficiency indices in beef cattle, especially ENU (body protein gain/N intake) and efficiency of metabolisable protein (MP) utilisation (body protein gain/MP intake). Although Δ¹5N obtained from different N pools in dairy cows were all negatively correlated with ENU, the highest correlation was found when Δ¹5N was calculated from plasma proteins. Δ¹5N showed no correlation with urea-N recycling or rumen NH3 absorption, but exhibited a strong correlation with liver urea synthesis and splanchnic amino acid metabolism, which points to a dominant role of splanchnic tissues in the present N isotopic fractionation study.

Effects of amino acid-derived luminal metabolites on the colonic epithelium and physiopathological consequences.

Depending on the amount of alimentary proteins, between 6 and 18 g nitrogenous material per day enter the large intestine lumen through the ileocaecal junction. This material is used as substrates by the flora resulting eventually in the presence of a complex mixture of metabolites including ammonia, hydrogen sulfide, short and branched-chain fatty acids, amines; phenolic, indolic and N-nitroso compounds. The beneficial versus deleterious effects of these compounds on the colonic epithelium depend on parameters such as their luminal concentrations, the duration of the colonic stasis, the detoxication capacity of epithelial cells in response to increase of metabolite concentrations, the cellular metabolic utilization of these metabolites as well as their effects on colonocyte intermediary and oxidative metabolism. Furthermore, the effects of metabolites on electrolyte movements through the colonic epithelium must as well be taken into consideration for such an evaluation. The situation is further complicated by the fact that other non-nitrogenous compounds are believed to interfere with these various phenomenons. Finally, the pathological consequences of the presence of excessive concentrations of these compounds are related to the short- and, most important, long-term effects of these compounds on the rapid colonic epithelium renewing and homeostasis.

Relationship between efficiency of nitrogen utilization and isotopic nitrogen fractionation in dairy cows: contribution of digestion v. metabolism?

Animal tissues are naturally 15N enriched relative to their diet and the extent of this difference (Δ15Nanimal-diet) has been correlated to the efficiency of N assimilation in different species. The rationale is that transamination and deamination enzymes, involved in amino acid metabolism are likely to preferentially convert amino groups containing 14N over 15N. However, in ruminants the contribution of rumen bacterial metabolism relative to animal tissues metabolism to naturally enrich animal proteins in terms of 15N has been not assessed yet. The objective of this study was to assess the impact of rumen and digestion processes on the relationship between Δ15Nanimal-diet and efficiency of N utilization for milk protein yield (milk N efficiency (MNE); milk N yield/N intake) as well as the relationship between the 15N natural abundance of rumen bacteria and the efficiency of N use at the rumen level. Solid- and liquid-associated rumen bacteria, duodenal digesta, feces and plasma proteins were obtained (n=16) from four lactating Holstein cows fed four different diets formulated at two metabolizable protein supplies (80% v. 110% of protein requirements) crossed by two different dietary energy source (diets rich in starch v. fiber). We measured the isotopic N fractionation between animal and diet (Δ15Nanimal-diet) in these different body pools. The Δ15Nanimal-diet was negatively correlated with MNE when measured in solid-associated rumen bacteria, duodenal digesta, feces and plasma proteins, with the strongest correlation found for the latter. However, our results showed a very weak 15N enrichment of duodenal digesta (Δ15Nduodenal digesta-diet mean value=0.42) compared with that observed in plasma proteins (Δ15Nplasma protein-diet mean value=2.41). These data support the idea that most of the isotopic N fractionation observed in ruminant proteins (Δ15Nplasma protein-diet) has a metabolic origin with very little direct impact of the overall digestion process on the existing relationship between Δ15Nplasma protein-diet and MNE. The 15N natural abundance of rumen bacteria was not related to either rumen N efficiency (microbial N/available N) or digestive N efficiency (metabolizable protein supply/CP intake), but showing a modest positive correlation with rumen ammonia concentration. When using diets not exceeding recommended protein levels, the contribution of rumen bacteria and digestion to the isotopic N fractionation between animal proteins and diet is low. In our conditions, most of the isotopic N fractionation (Δ15Nplasma protein-diet) could have a metabolic origin, but more studies are warranted to confirm this point with different diets and approaches.

Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells.

The gut absorption of proanthocyanidins (PAs) and of the related (+)-catechin monomer was investigated with colonic carcinoma (Caco-2) cells of a human origin, grown in monolayers on permeable filters. Permeability of various radiolabeled PAs differing in their molecular weight was compared with that of the radiolabeled (+)-catechin. No toxicity was observed at PA concentrations up to the physiological concentration of 1 mM. (+)-Catechin and PA dimer and trimer had similar permeability coefficients (P(app) = 0.9-2.0 x 10(-6) cm s(-1)) close to that of mannitol, a marker of paracellular transport. Paracellular transport was also indicated by the increase of absorption after reduction of the transepithelial electric resistance through calcium ion removal. In contrast, permeability of a PA polymer with an average polymerization degree of 6 (molecular weight 1,740) was approximately 10 times lower (P(app) = 0.10 +/- 0.04 x 10(-6) cm s(-1)). PAs, particularly the most astringent PA polymer, were also adsorbed on the epithelial cells. These results suggest that PA dimers and trimers could be absorbed in vivo and that polymer bioavailability is limited to the gut lumen.

Gastroileal nitrogen and electrolyte movements after bovine milk ingestion in humans.

Gastric emptying and flow rates of nitrogen and electrolytes (Na+, K+, Cl-, Mg2+, Ca2+) were studied in humans after bovine milk ingestion. With water as the control, intestinal effluents were collected after meal ingestion at the beginning of the jejunum or in the distal ileum. The flow rate of the effluent peaked in the first 40-min period after meal ingestion and returned to the initial amount within 100 min. After water ingestion the quantity of nitrogen recovered in the digesta remained unchanged both in the jejunum and in the ileum during the test period. After milk ingestion the nitrogen concentration in the jejunal digesta peaked in the first 20 min. Forty-two percent of milk nitrogen was absorbed before the jejunum and 93% was absorbed before the end of the ileum. These results showed that for the completion of the absorption of dietary proteins such as milk proteins, the lower part of the intestine is necessary.

Metabolism of enterostatin in rat intestine, brain membranes, and serum: differential involvement of proline-specific peptidases.

The degradation of enterostatin (VPDPR), a potent inhibitor of food intake, by intestinal brush-border membranes, brain membranes, and rat serum has been investigated in the presence of specific inhibitors. Hydrolysis by intestinal membranes was found to be 10 and 100 times faster than in serum and brain membranes, respectively. Enterostatin hydrolysis by intestinal and brain membranes involves the removal of C-terminal arginine by carboxypeptidase P, leading to the production of des-Arg-enterostatin, and the splitting of the Pro2-Asp3 bond by dipeptidyl aminopeptidase IV (DPP IV). A small amount of the potent anorectic peptide Pro2-Asp3-Pro4 was released during hydrolysis of des-Arg-enterostatin by brain membranes and rat serum. In rat serum, enterostatin degradation was mainly due to DPP IV.

Peptide fragments released from Phe-caseinomacropeptide in vivo in the rat.

The aim of this study was to investigate the pharmacokinetics of bovine Phe-caseinomacropeptide (Phe-CMP) in the rat after oral administration. This polypeptide was monophosphorylated and mainly nonglycosylated: Phe-CMP-1P. During gastrointestinal digestion and absorption, Phe-CMP-1P was degraded. Intact Phe-CMP-1P and CMP-1P were rapidly released from the stomach. In contrast, partial hydrolysis by pancreatic enzymes was observed. In vitro hydrolysis by brush-border membrane vesicles also indicated that the peptide was degraded. In the blood, "CMP-immunoreactive material" appeared rapidly, reaching a maximum level of 5.5 microg/ml at 60 min.

The in vitro intestinal absorption of enterostatin is limited by brush-border membrane peptidases.

The intestinal metabolism and absorption of enterostatin was studied using brush-border membrane vesicles and an in vitro model of intestinal segments from rabbit ileum mounted in Sweetana-Grass diffusion chamber. Hydrolysis of enterostatin was observed with both epithelial sheets and brush-border membranes. The main metabolite was found to be des-arginine-enterostatin. Dipeptidylpeptidase IV was found to play a minor role in enterostatin degradation, whereas carboxypeptidase P activity accounted for the initial step of peptide hydrolysis. More than 50% of the amount of enterostatin added to the mucosal compartment of the Sweetana-Grass diffusion chamber was degraded after 30 min. Enterostatin was mainly absorbed as degradation products but a small transepithelial passage of des-arginine-enterostatin and immunoreactive enterostatin was also detected. Although immunoreactive enterostatin exhibits a low apparent permeability coefficient in rabbit ileum, the luminal production of this peptide may be of physiological importance in the control of appetite.

The inhibition of intestinal dipeptidylaminopeptidase-IV promotes the absorption of enterostatin and des-arginine-enterostatin across rat jejunum in vitro.

Transport of enterostatin (VPDPR) across rat jejunum was investigated using Grass-Sweetana diffusion chambers. The rate of absorption of enterostatin and its metabolites were studied in absence and in presence of diisopropylfluorophosphate (DFP), a serine protease inhibitor. An extensive hydrolysis of enterostatin was observed during incubation with brush border membranes and when exposed to the mucosal side of the jejunal epithelium. No accumulation of enterostatin occurred in serosal tissue. Addition of DFP delayed enterostatin disappearance and abolished des-arg-enterostatin degradation. Under these conditions, a low amount of enterostatin was able to cross the epithelium intact. Moreover, a substantial amount of des-arg-enterostatin is absorbed across the jejunal epithelium, probably through passive diffusion. Thus, a decreased metabolic activity increased the absorption of a tetrapeptide (VPDP). Dipeptidylaminopeptidase IV, remains a limiting step in transfer of intact enterostatin and its metabolite des-arg-enterostatin across intestinal wall.

Evidence for a passive diffusion mechanism for sparfloxacin uptake at the brush-border membrane of the human intestinal cell-line Caco-2.

The oral uptake of the new fluoroquinolone sparfloxacin was evaluated in the human epithelial cell line Caco-2 that possesses intestinal enterocyte-like properties when cultured in vitro. The uptake of [14C]-sparfloxacin across the apical membrane of Caco-2 cell monolayers was rapid and similar at 25 and 37 degrees C. The initial rate of sparfloxacin uptake was not saturable in the 1-200 microM range and was unaffected by metabolic inhibitors (depletion of ATP store or ouabain), indicating that uptake was energy-independent. The absence of competition with other fluoroquinolones or aminocephalosporins showed that the absorption of sparfloxacin did not involved the H+-coupled dipeptide transport system. Our findings suggest that the apical uptake of sparfloxacin by Caco-2 cells mainly involves diffusion, a finding that is in agreement with the high lipophilicity of sparfloxacin. The intracellular-to-extracellular concentration ratio of approximately 14 after 60 min of incubation suggests the existence of important binding of sparfloxacin to cell components.

Dietary protein and cardiovascular risk.

The relations between dietary protein and cardiovascular risk were first considered through their impact on blood cholesterol. Half a century after the first reports of an hypocholesterolemic effect of plant proteins, this subject is still a mater of debate, notably because of the difficulty in distinguishing between an independent effect of proteins and that of phytochemicals present in proteins preparations. In addition, many questions still have to be answered as to how the proteins may affect cholesterol metabolism. This review also describes the recent advances in new areas of research that have recently gained attention. Dietary proteins may affect cardiovascular risk through their effect on homocysteine, glutathione and nitric oxide. Although most of the data now available are still inconclusive, incoming results on these topics may prove important to appraise the role that the amount and/or the nature of dietary proteins play in the onset of cardiovascular disease.

Proanthocyanidins and human health: systemic effects and local effects in the gut.

Proanthocyanidins share common properties with other polyphenols, in particular their reducing capacity and ability to chelate metal ions. However, their polymeric nature clearly makes them different. They have a high affinity for proteins and their absorption through the gut barrier is likely limited to the molecules of low polymerization degree and to the metabolites formed by the colonic microflora, as suggested by in vitro experiments. The nutritional significance of proanthocyanidins is discussed in relation to their physico-chemical properties and bioavailability.

Effect of chronic antigen exposure on growth and intestinal histamine content of sensitized rats.

The effects of chronic antigen feeding on systemically sensitized rats were investigated. Findings include a reduction of water and antigen intake in egg albumin (EA), sensitized rats receiving EA in their drinking water for an 8 day period, compared to that of sensitized rats fed bovine serum albumin and of naive rats. Feeding EA to sensitized animals also induced a decrease in daily weight gain. This decline did not seem to be a consequence of a decreased food intake, but might rather reflect a decreased water consumption and an alteration of nutrient absorption in the gut. Indeed, sensitized rats fed EA exhibited a significant increase in jejunal and ileal histamine content compared to control rats, which may indicate the development of an inflammatory reaction in the small intestinal mucosa. Intestinal troubles experienced because of this inflammatory reaction might explain the reduction of antigen and water intake observed in sensitized rats.

Nitrogen movements in the upper jejunum lumen in humans fed low amounts of casein or beta-lactoglobulin.

OBJECTIVES AND METHODS: To compare the progression of milk proteins in the upper part of the digestive tract, gastro-jejunal nitrogen movements were studied in 6 healthy human volunteers after beta-lactoglobulin and casein ingestion. 400 mL of water (control), purified beta-lactoglobulin (20 g/L) or casein (20 g/L), each adjusted to 25 microCi with 14C-polyethylene glycol, were given per os. Samples were collected in the stomach and 20 cm below the Treitz ligament every 20 min for 2 hours and measured for volume, osmolarity, ions and nitrogen content. RESULTS: The jejunal flow rate peaked in the 0-20 min period following water and beta-lactoglobulin ingestion, and in the 20-40 min period after casein ingestion. The gastric half-emptying time (T1/2 min) of the liquid phase was significantly different (P < 0.05) for water (12.1 +/- 0.8), beta-lactoglobulin (14.5 +/- 3.3) and casein (26.5 +/- 9.3). Before ingestion of the test meals, the basal rate of nitrogen was 9.14 +/- 4.09 mmol/h in the jejunum. The total nitrogen content in the jejunum peaked significantly in the 0-20 min period after beta-lactoglobulin ingestion and the 20-40 min period after casein ingestion. The apparent gastro-jejunal protein absorption values were 63% for casein and 66% for beta-lactoglobulin in the 120 min period. CONCLUSIONS: These results show that beta-lactoglobulin and casein behave differently in the upper part of the digestive tract due to different gastric emptying rates.

[Cryptosporidium parvum: functional study of the intestinal malabsorption syndrome]. / Cryptosporidium parvum: étude fonctionnelle du syndrome de malabsorption intestinale.

Cryptosporidiosis is an important cause of diarrhea associated with growth retardation in children and severe malnutrition in immunocompromised patients. The pathophysiology is poorly understood. In the suckling rat model, we show that C. parvum infection impairs net electrogenic transport across the ileal mucosa without involvement of prostaglandins, as well as trans- and paracellular permeability and leucine and glutamate absorption. These results provide evidence for the development of an intestinal malabsorptive syndrome during cryptosporidiosis. Unspecific process such as villous atrophy and inflammatory cytokines secretion should be regarded as possible mediators of this syndrome. However, specific mechanisms have to be considered since C. parvum induces a rearrangement of the host enterocyte cytoskeleton which might impaired intracellular trafficking thus reducing the membrane expression of nutrient transporters. Infection and malnutrition are known to be tightly associated, making each other worse. As no specific efficient therapy exists, cryptosporidiosis-induced malnutrition must be taken into account when establishing therapeutic scheme.

N-3 fatty acids improve body composition and insulin sensitivity during energy restriction in the rat.

The hypothesis that n-3 polyunsaturated fatty acids (PUFA) could contribute to maintain muscle mass during energy restriction aiming to weight loss was tested in the rat, with special attention paid to insulin signalling. After 10 weeks on a diet rich in lipids and sucrose, male rats were energy restricted and fed diets rich in 18:1 n-9 (OLE), 18:3 n-3 (ALA) or n-3 long-chain (LC, >18 carbons) PUFA. After 4 weeks, they were killed after an insulin injection. Red blood cells, liver, and Gastrocnemius muscle were enriched in ALA in the ALA group, and in LC-PUFA in the ALA and LC groups. The LC diet resulted in a higher weight loss, without negative impact on the muscle weight. In parallel, hepatic phosphorylation of insulin receptor and IRS1 was the highest in this group. This suggests that the trend we observed in the preservation of protein homeostasis in the LC group is mediated, at least partly, by an enhancement of the early steps of insulin signalling resulting from cell membrane enrichment in n-3 PUFA.

In vitro transport of beta-lactoglobulin across the jejunum of lactose-fed rats.

Changes in protein absorption through the intestinal mucosa occur with aging and might reflect modifications in enterocyte membrane characteristics. We have observed that bovine beta-lactoglobulin (beta-LG) was efficiently transported across the small intestine of adult rats in vitro and that 12-16% of the absorbed protein was recovered intact or as large hydrophobic peptides. Ten percent lactose-feeding resulted in decreased tissue conductance and significantly reduced (-58%, P less than 0.05) beta-LG transport across rat small intestinal mucosa. The amounts of beta-LG absorbed, either as amino acids and peptides or as intact protein, were reduced to the same extent. Therefore, the effect of lactose feeding might be related to a decrease in protein endocytosis at the brush-border level, rather than to reduced protein transport across tight junctions.

Effect of serum on the metabolism of exogenous arachidonic acid by phagocytic cells of the mouse thymic reticulum.

Phagocytic cells derived from the mouse thymic reticulum (P-TR) were used to study the effect of serum on the metabolism of arachidonic acid (AA). After labeling with (14C)-AA in the presence of 10% serum, we failed to detect in the culture medium the presence of significant amounts of radiolabeled prostaglandins. In contrast, when the labeling period was carried out in serum-free medium, we observed the secretion of both cyclooxygenase and lipoxygenase derivatives. In addition, the pattern of arachidonate incorporation into cell lipids was different in the two culture conditions. In the presence of serum, the great majority of the radioactivity was found associated with phospholipids, whereas in serum-free medium, almost 50% of the incorporated fatty acid was associated with triglycerides. Since serum albumin is known to play a major role in the control of fatty acid uptake, we have studied the effect of the addition of 2% BSA to cells prelabeled in the absence of serum. This treatment switches the patterns of metabolite release and lipid labeling towards those of serum-treated cells. In addition, we showed that the effects of glucocorticoids on AA release differ markedly according to the composition of the culture medium.