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.

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.

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.

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.

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.

Evidence for the absence of an intestinal adaptive mechanism to compensate for C. parvum-induced amino acid malabsorption in suckling rats.

In order to assess the impact of Cryptosporidium parvum on host intestinal physiology, we investigated absorption of the two principal amino acids in dam's milk (leucine, glutamate), using Ussing chambers and RT-PCR analyses. Experiments were performed in both heavily (ileum) and mildly (duodenum) infected segments of the small intestine at the peak of infection [day 8 post-infection (PI)] and after spontaneous clearance of the parasite (day 17 PI). At day 8 PI, amino acid fluxes across the mucosa were decreased throughout the small intestine (P<0.01) and EAAT3 mRNA expression was reduced ( from -49% to -28%). At day 17 PI, leucine and glutamate fluxes were normalized but the decrease in EAAT3 mRNA levels persisted (from -31% to -46%). Our results demonstrate that cryptosporidiosis induces major amino acid malabsorption involving the entire small intestine which is not counterbalanced by any up-regulation, even after spontaneous clearance of the parasite.

Intestinal peptide transporter PepT1 is over-expressed during acute cryptosporidiosis in suckling rats as a result of both malnutrition and experimental parasite infection.

Cryptosporidium parvuminfection induces amino acid malnutrition leading to growth retardation in children. Owing to the nutritional efficiency of peptides compared to free amino acids and the resistance of the di-tripeptide transporter PepT1 to mucosal injury, we analyzed the intestinal expression of PepT1 during experimental acute cryptosporidiosis in suckling rats from day 4 to day 50. PepT1 mRNA levels were increased at the peak of infection (day 10) all along the small intestine and normalized after spontaneous clearance of the parasite (day 21). Immunolocalization of PepT1 showed that its expression was maintained in the brush border membrane of enterocytes in infected rats from day 4 to day 50 all along the small intestine. Our results suggest a transcriptional up-regulation during acute cryptosporidiosis in response to both C. parvum-induced malnutrition and parasite implantation. As no treatment is available, a semi-elemental diet should be considered part of the treatment of cryptosporidiosis.

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.

Impairment of amino-acid absorption in suckling rats infected with Cryptosporidium parvum.

In the present study. we explored the nutritional consequences of cryptosporidiosis. In order to ascertain the direct responsibility of C. parvum for impairment of staturoponderal development observed during the infection in neonatal animals, we investigated the absorption of two major components of the total amino acids in dam's milk (leucine and glutamate) across the ileal mucosa. The infection resulted in significant (47% and 34%, respectively) reductions in leucine and glutamate fluxes (P<0.01). Moreover, the leucine aminopeptidase and alkaline phosphatase activities were reduced in the infected ileal mucosa. Interestingly, the reduction in weight gain, which began at day 6 post-infection (PI), persisted until day 20 PI, although no cryptosporidia were detected in the ileal mucosa after day 12 PI. We thus provide evidence that the malabsorption of amino acids during cryptosporidiosis contributes to impairing the development of neonatal animals, with consequences that persist beyond eradication of the parasite.

Energy nutrients modulate the splanchnic sequestration of dietary nitrogen in humans: a compartmental analysis.

We used a previously developed compartmental model to assess the postprandial distribution and metabolism of dietary nitrogen (N) in the splanchnic and peripheral areas after the ingestion of a single meal containing milk protein either alone (MP) or with additional sucrose (SMP) or fat (FMP). The addition of fat was predicted to enhance splanchnic dietary N anabolism only transiently, without significantly affecting the global kinetics of splanchnic retention and peripheral uptake. In contrast, the addition of sucrose, which induced hyperinsulinemia, was predicted to enhance dietary N retention and anabolism in the splanchnic bed, thus leading to reduced peripheral dietary amino acid availability and anabolism. The incorporation of dietary N into splanchnic proteins was thus predicted to reach 18, 24, and 35% of ingested N 8 h after MP, FMP, and SMP, respectively. Such a model provides insight into the dynamics of the system in the nonsteady postprandial state and constitutes a useful, explanatory tool to determine the region-specific utilization of dietary N under different nutritional conditions.

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.

Metabolic evidence for adaptation to a high protein diet in rats.

This study was designed to assess the effects of long-term adaptation to a high protein diet on energy intake, body weight gain, body composition and splanchnic metabolic indicators in rats. For this purpose, adult male Wistar rats were fed either a 50 g/100 g dry matter (DM) protein diet (P50 group) or a 14 g/100 g DM protein diet (P14 group) for 21 d. These two groups were compared with a P14 pair-fed (P14-pf) group that consumed the same daily energy as the P50 group. The energy intake of the P50 group was 16 +/- 1% less than that of the P14 group (P < 0.05), and the P50 group had significantly lower body weight. The P50 group had significantly less adipose tissue compared with both P14 and P14-pf rats. The activities of the brush border membrane enzymes, neutral aminopeptidase and gamma-glutamyl transferase, were significantly higher in the P50 group than in the P14 rats. Similarly, the activities of alanine aminotransferase, arginase and serine dehydratase were significantly higher in the liver of P50 rats compared with P14 rats. Both amino acid transporter system A and X(A,G-) activities, measured in freshly isolated hepatocytes, were significantly higher in the P50 group (8- and 1.5-fold, P < 0.05, respectively) compared with the P14 group. The 1.5-fold increase in the steady-state activity of X(A,G-) was accompanied by a doubling of EAAT2 mRNA, involved in the system X(A,G-). This study provides confirmation that specific biochemical and molecular adaptive processes of the splanchnic area are involved in the response to variations in the protein content of the diet.

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.

[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.

A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet.

The postprandial fixation of dietary nitrogen in splanchnic and peripheral tissues as well as its dynamic transfer to the nitrogen pools of the body were quantified in rats subjected to an acute augmentation of dietary protein. For this purpose, we traced the dietary protein and studied the immediate fate of exogenous nitrogen in many tissues and biological fluids. Rats were adapted to a diet providing an adequate protein level (14 g/100 g), and then fed a meal containing either 0.42 g (Group A) or 1.50 g (Group H) of [(15)N]-labeled milk protein. The amounts of exogenous nitrogen transferred to urea (0.32 +/- 0.04 vs. 2.46 +/- 0.25 mmol, respectively), incorporated in splanchnic (0.41 +/- 0.02 vs. 0.87 +/- 0.10 mmol) and peripheral (1.65 +/- 0.84 vs. 2.36 +/- 0.49 mmol) tissue protein were higher in group H than in group A. Individual plasma amino acids (AA) [(15)N]-enrichments showed that AA respond differentially to an acute augmentation of dietary intake. This work provides new descriptive and quantitative information on the metabolic fate of dietary nitrogen in the postprandial state. It highlights the higher integration of a surplus of dietary nitrogen in the tissues even if it is rapidly limited by saturation of the protein synthesis capacities. The main metabolic response remains the stimulation of AA degradation, leading to a large rise in urea production. However, both anabolic and catabolic systems are exceeded, resulting in an elevation of peripheral AA and negative feedback on the gastric emptying rate.

EAAT1 is involved in transport of L-glutamate during differentiation of the Caco-2 cell line.

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism of L-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity, D-aspartate-sensitive L-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na(+) dependent, with a Hill coefficient of 2. 9 +/- 0.3, suggesting a 3 Na(+)-to-1 glutamate stoichiometry and corresponding to the well-characterized X(A,G)(-) system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B(0) and y(+), which have previously been reported to be downregulated when Caco-2 cells stop proliferating, L-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

Compartmental modeling of postprandial dietary nitrogen distribution in humans.

A linear 11-compartment model was developed to describe and simulate the postprandial distribution of dietary nitrogen. The values of its 15 constant diffusion coefficients were estimated from the experimental measurement of (15)N nitrogen kinetics in the intestine, blood, and urine after the oral administration of (15)N-labeled milk protein in humans. Model structure development, parameter estimation, and sensibility analysis were achieved using SAAM II and SIMUSOLV softwares. The model was validated at each stage of its development by testing successively its a priori and a posteriori identifiability. The model predicted that, 8 h after a meal, the dietary nitrogen retained in the body comprised 28% free amino acids and 72% protein, approximately 30% being recovered in the splanchnic bed vs. 70% in the peripheral area. Twelve hours after the meal, these values had decreased to 18 and 23% for the free amino acid fraction and splanchnic nitrogen, respectively. Such a model constitutes a useful, explanatory tool to describe the processes involved in the metabolic utilization of dietary proteins.

Protein metabolism and the gut.

This paper reviews the recent literature concerning the importance of the gut in extraintestinal protein metabolism. A growing body of evidence suggests that the gut modulates amino acid flux and inter-organ relationships in various metabolic states. This may be particularly true during the absorptive period, when the gut: (1) controls amino acid absorption; (2) may modulate catabolism and uptake for synthesis of absorbed amino acid; and (3) consequently influences the availability of liver and extrasplanchnic amino acids, as well as their pattern and kinetics through portal flow delivery.

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.

Cryptosporidium parvum infection in suckling rats: impairment of mucosal permeability and Na(+)-glucose cotransport.

Na(+)-glucose transport and transepithelial permeability were investigated during symptomatic acute cryptosporidiosis in newborn rats. The infection resulted in a significant (P < 0.01) decrease in the ileal short-circuit current and a nonsignificant fall in the transepithelial potential difference and conductance. In glucose-stimulated conditions, the rise in ileal short-circuit current and transepithelial permeability were significantly lower in Cryptosporidium parvum-infected rats than in controls (delta Isc = 3.24 +/- 1.21 microA.cm-2 vs delta Isc = 5.09 +/- 2.23 microA.cm-2 in infected and control animals, respectively; P < 0.001; delta PD = -0.35 +/- 0.13 mV vs delta PD = -0.44 +/- 0.14 mV for infected and control animals, respectively; P < 0.01). Electrical parameters were not affected by addition of the cyclooxygenase inhibitor indomethacin in either Cryptosporidium-infected newborn rats or controls. Horseradish peroxidase and mannitol flux studies demonstrated a significant decrease (P < 0.05) in transepithelial molecular permeability in infected enterocyte rats, HRP flux = 380, range 68-5570 ng.cm-2, and mannitol flux = 1.06, range, 0.34-1.44%.cm-2.min-1, compared with controls rats, HRP flux = 4446 range, 1121-124,363 ng.cm-2, and mannitol flux = 1.99, range, 0.57-5.09%.cm-2.min-1; P < 0.05. These effects could originate from C. parvum-induced alteration of intracellular trafficking of pinocytosis vesicles and therefore account for the decrease in permeability to solute and macromolecules, together with impaired transcellular nutrient transport, in suckling rats.