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Article

A High-Protein Meal Exceeds Anabolic and Catabolic Capacities in Rats Adapted to a Normal Protein Diet

^* Institut National de la Recherche Agronomique, Unité de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique de Paris–Grignon, 16 Rue Claude Bernard, F-75005 Paris, France; ^** German Institute of Human Nutrition, Arthur-Scheunert-Allee 114–116, D-14558 Bergholz–Rehbruecke, Germany

¹To whom correspondence should be addressed at INRA UPNCA, INA–PG, 16 Rue Claude Bernard, 75231 Paris Cedex 05, France.

	ABSTRACT

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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 [¹⁵N]-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) [¹⁵N]-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.

KEY WORDS: • rats • protein metabolism • high-protein meal • [¹⁵N]

	INTRODUCTION

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The body protein content remains relatively constant throughout most of adult life, although there are broad qualitative and quantitative variations in the daily dietary protein intake. Indeed, protein and nitrogen homeostasis and nitrogen balance are achieved through a complex equilibrium between protein intake and protein and amino acid (AA)² metabolism, which includes whole body protein turnover, AA oxidation, urea production and nitrogen excretion during the fasting and fed periods of the day. The metabolism and deposition of dietary nitrogen during the postprandial phase are likely to be critical steps in response to acute variations in protein intake, so as to sustain AA concentrations in the blood and tissues at physiological levels.

Depending on protein intake, protein and AA oxidation and the disposal of nitrogen via the urea cycle respond more or less rapidly to intake of a protein meal by modulating the activity of catabolic enzymes such as glutaminase, and subsequently the tissue AA concentration (Cohen 1981 , Ewart and Brosnan 1993 , Harper et al. 1984 ). The incorporation of AA into body protein is also modulated in response to changes in protein intake, but the postprandial first pass net uptake of dietary AA in different tissues is not clearly established (Garlick et al. 1999 ). The splanchnic tissues have been shown to play a prominent role in the first-pass metabolism of dietary AA (Stoll et al. 1998a , 1998b and 1999 ). Depending on the AA, between 20 and 50% of dietary AA are taken up by the small intestine following meal ingestion (Windmueller and Spaeth 1975 ). In the case of glutamic acid, >90% are removed by the portal-drained viscera, suggesting that most of the glutamic acid in peripheral tissues must be generated by de novo synthesis (Reeds et al. 1996 ). A rise in plasma AA concentrations may also be observed following a high-protein meal associated with an anorexic response (Peters and Harper 1985 , Semon et al. 1988 ).

Despite their important role in the short-term response to variations in protein intake, the capacities of the first-pass distribution and postprandial metabolism of dietary protein and AA nitrogen to react quickly to different levels of protein intake are still poorly understood, especially due to the lack of studies focusing on dietary compounds. The data reported in this study are the first to quantify the incorporation of dietary nitrogen in the splanchnic and peripheral tissues as well as its dynamic transfer to the nitrogen pools of the body. They allow assessment of the postprandial anabolic and catabolic capacities of the organs to respond acutely to a rise of protein intake. For this purpose, the immediate fate of dietary nitrogen was traced in several splanchnic (intestine, liver) and peripheral (muscle, kidney) tissues as well as in the protein, free individual AA and urea of plasma after the intake of a single mixed meal containing either 0.42 g (Meal A) or 1.50 g (Meal H) of [¹⁵N]-labeled milk protein in rats adapted to an adequate 14% protein diet.

	MATERIALS AND METHODS

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[¹⁵N]-labeled compounds.

[¹⁵N]-labeled milk was produced at the Institut National Agronomique experimental farm, in Grignon with the help of Dr. P. Schmidely (Department of Animal Sciences, INA-PG, Grignon, France). Milk was [¹⁵N]-labeled by giving 50 g/d of ([¹⁵N]H₄)₂SO₄ (10 atom % isotope enrichment; Euriso-top, Saint Aubin, France) via the oral route to a lactating cow for 11 d. The milk collected each day was pooled and then defatted. Proteins were concentrated by diafiltration (UFP 1.1 m² IRIS 3065 Rhône Poulenc 40 kDa membranes) and the concentrated proteins lyophilized. The isotopic enrichment of concentrated milk proteins was 0.4535 atom % ¹⁵N (AP). Labeling of the protein was almost uniform among the different AA ranging only from 0.4350 AP for histidine and proline to 0.4680 AP for lysine (Table 1 ).

Experiments were carried out in accordance with the recommendations of the French Committee for Animal Care. Male Wistar rats (n = 76; Harlan, France), weighing 192 ± 8 g at the beginning of the experiment, were housed individually in stainless steel wire cages in a room with a controlled temperature (23 ± 2°C) and a 12-h light-dark cycle (light 2030–0830). They were adapted for 14 d to an AIN-93M modified diet (Reeves et al. 1993 ). Instead of casein and cysteine, this diet contained 140 g of total milk protein per kg of feed (Table 2 ). The diet was moistened (water/powdered diet, 1:1) to prevent spillage. The rats had free access to water throughout the experimental period. A special daily feeding schedule was used under which rats were given three meals per day. Between 0830 and 0845 h, 3 g of the diet were given to the rats. Between 1330 and 1430 h and between 1830 and 1930 h, rats had free access to the food. This schedule was chosen to adapt the rats to prompt consumption of the diet, while ensuring an adequate level of daily food intake (20.9 ± 0.2 g/d dry food). On the morning of d 15 (0830–0845), the 3-g meal contained either 0.42 g (Meal A, n = 33) or 1.50 g (Meal H, n = 36) of [¹⁵N]-labeled milk protein. Meal A contained the same amount of [¹⁵N]-labeled protein as that consumed during the adaptation period, whereas Meal H represented a 3.6-fold increase of dietary protein compared to the adaptation meals. The compositions of experimental meals are described in Table 2 . Seven rats were not given any food and immediately anesthetized (food-deprived group).

Absorbent paper was placed under the cages to collect the urine excreted on the day of the experiment. At zero, 1, 2, 3, 4 or 5 h (n = 7 at each time point except for meal A at 4 and 5 h where n = 6 and meal H at 3 h where n = 8) after the [¹⁵N]-labeled meal, the rats were anesthetized with sodium pentobarbital [13.6 mg/100 g body weight (BW)]. They were also injected with 5000 UI heparin (Laboratoires Leo, Saint-Quentin en Yvelines, France). The abdomen was then opened and blood was removed from the cavity after rupture of both the abdominal aorta and vena cava. The blood was placed in a glass tube and centrifuged for 15 min at 3000 x g (4°C). The plasma was collected and stored at -80°C until analysis. The bladder was removed and emptied of urine. The absorbent paper was rinsed with distilled water and the eluate pooled with the urine from the bladder. The urine was stored at -80°C until analysis. Stomach, gut and cecum digesta, liver, small intestine and colon mucosa, one kidney and one gastrocnemian muscle were sampled, weighed and stored at -80°C until analysis.

Analytical methods.

Levels of urea in blood and urine were determined using an enzymatic method (urease-glutamate dehydrogenase; HYCEL kit, Le Rheu, France) on a Mascott Plus robot (HYCEL, Le Rheu, France). Urinary urea was extracted by cation exchange chromatography on Dowex resin (Dowex AG50X8; Biorad, Ivry sur Seine, France) as previously described (Gaudichon et al. 1999 ) and stored at 4°C until isotopic determination. Plasma urea was also extracted using the same procedure, after deproteinization with 1 mol/L HCl and neutralization with 0.1 mol/L NaH₂PO₄.

For AA analysis, plasma was first deproteinized with sulfosalicylic acid (50 g/L), stored at 4°C for 1 h and then centrifuged at 3000 x g (4°C) for 15 min. The supernatant was dried and resuspended in a lithium citrate buffer (pH 2.2) for analysis. Plasma AA concentrations were determined using postcolumn ninhydrin detection on an automatic Alpha Plus analyzer (Pharmacia LKB; Biochrom, Cambridge, United Kingdom). The pellet, containing plasma proteins, was lyophilized.

[¹⁵N]-enrichment of individual plasma AA was measured using gas chromatography-combustion-isotopic ratio mass spectrometry (GC-C-IRMS) analysis. Plasma (0.6 mL) was centrifuged at 3000 x g for 1 min and acidified with 1 mol/L HCl. AA were extracted on individual columns filled with Dowex AG50X8 resin (mesh 100–200). After the sample solution had entered the resin, the column was washed with 8 mL distilled water and the effluent discarded. Plasma AA were then immediately eluted with 3 mL 4 mol/L NH₄OH and 1 mL distilled water. The eluate was dried and the AA derivatized to N-pivaloyl-i-propyl (NPP) AA esters, as previously described (Metges and Petzke 1997 ). Briefly, the AA were treated with 1 mL of a thionylchloride and i-propanol solution. The mixture was heated for 30 min at 110°C and then dried in a gentle stream of nitrogen at 60°C and redissolved in pyridine (100 µL). After the addition of pivaloylchloride (100 µL), each solution was acylated for 30 min at 60°C, cooled and then 2 mL of dichloromethane were added. The solution was then passed through a silica gel column and the eluate dried in a gentle nitrogen stream at room temperature. The vials were covered and the derivatives kept refrigerated until GC-C-IRMS analysis.

Tissue samples were crushed in 4 vol of 9 g/L NaCl. Protein was precipitated with trichloroacetic acid (TCA, 612 mmol/L, final concentration). After 15 min at room temperature, samples were centrifuged (15 min, 3000 x g, 4°C), and the supernatant (containing free AA and small peptides) was frozen and then dried. The pellet (containing protein) was washed once in 9 g/L NaCl and then frozen and lyophilized.

Isotopic determinations.

Before the isotopic determination of [¹⁵N]-enrichment, resins were eluted with 2.5 mmol/L KH₂SO₄. [¹⁵N]-enrichment was measured in urinary and blood urea as well as in protein (P) and nonprotein (NP) fractions of tissues, in plasma proteins and in gastrointestinal digesta using IRMS (Optima; Fisons Instruments, Manchester, United Kingdom) coupled to an elemental analyzer (NA 1500 series 2; Fisons Instruments). Calibrated nitrogen gas was used as the ¹⁵N/¹⁴N reference. The AP = ¹⁵N/(¹⁴N + ¹⁵N) and the atom % excess (APE = AP - natural enrichment of the sample) were then calculated. Total nitrogen in the P and NP fractions of tissues (i.e., gut and colon mucosa, liver, muscle, kidney) was measured with an elemental nitrogen analyzer with atropina (Carlo Erba Intruments, Fisons, Arcueil, France) as a standard, as previously described (Gausserès et al. 1997 ).

Prior to GC-C-IRMS analysis, the derivatized AA were resuspended in 50 µL ethylacetate. Analyses were performed using a Finnigan delta S Isotope Ratio Mass Spectrometer (Finnigan MAT, Bremen, Germany) coupled on-line with a gas chromatograph (GC, HP 5890; Hewlett Packard, Walbronn, Germany), as previously described (Metges and Petzke 1997 ). A combustion interface allows the production and purification of N₂ gas from GC-separated compounds to enter the isotope ratio mass spectrometer. An Ultra 2 capillary column (50 m; Hewlett Packard) was used to separate the AA. The carrier gas was He. Introduction of a standard N₂ gas (known isotopic composition) was used for calibration.

Calculations and statistics.

The exogenous N present in samples (N_exo, mmol) was calculated as follows:

where N_tot is the amount of total N in the sample, APE_s and APE_m the [¹⁵N]-enrichment excess of the sample and the meal, respectively.

The exogenous nitrogen present in the urea body pool (N_exo-urea, mmol) was calculated according to the formula:

where BW is the body weight, C_urea the concentration of urea in the plasma and APE_ureas and APE_m the [¹⁵N]-enrichment excess of the plasma urea sample and the meal, respectively. The mean percentage of body water in the rat and the mean percentage of water in plasma are 67 and 92%, respectively (Sharp and La Regina 1998 ). The total transfer of dietary nitrogen to urea was calculated as the sum of the exogenous nitrogen excreted in urinary urea and the exogenous nitrogen present in the body urea pool.

Body plasma volume was calculated assuming that there are 3.5 mL of plasma per 100 g of BW (Waynforth 1980 ).

Body muscle weight (BMW, g) was evaluated as follows:

where 45% is the mean percentage of muscle in rats and BW the body weight (Even, P. C., INRA UPNCA, personal communication).

The percentage of ingested nitrogen (P_N, %) recovered in the different tissues was calculated as follows:

where N_{exo tot} is the total amount of dietary nitrogen recovered in the tissue and N_m the total amount of exogenous N ingested from the meal.

The plasma [¹⁵N]-AA data were expressed in terms of cumulative dose (µmol/L x 5 h):

The results were expressed as means with their standard errors. Differences between meal groups as well as comparisons with the food-deprived group (i.e., basal values) were tested using ANOVA and (Proc GLM, SAS version 6.11; Cary, NC). Differences of P < 0.05 were considered significant.

	RESULTS

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Nitrogen transfer to urea and unabsorbed nitrogen.

Values for body urea nitrogen and urea nitrogen excretion in urine were determined over the 5-h period following the ingestion of meals (Fig. 1 ). Body urea nitrogen increased progressively in rats fed meal H, whereas it remained stable for 3 h in those fed Meal A and then decreased. In urine, the excretion of urea nitrogen was higher in rats given Meal H than in those given Meal A. In both plasma and urine, the [¹⁵N]-enrichment of urea was significantly higher in rats fed Meal H than in those given Meal A (P < 0.05) (Fig. 2 ). As an example, 5 h after Meal H, plasma urea [¹⁵N]-enrichment reached 0.0327 ± 0.0020 APE, while it was only 0.0075 ± 0.0008 APE after Meal A. Moreover, the kinetics differed between the two groups, since enrichment rapidly stabilized in rats given Meal A, whereas it increased for a longer time in rats ingesting Meal H, especially for plasma urea.

View larger version (22K): [in this window] [in a new window]   Figure 1. Body urea nitrogen and urinary urea nitrogen excretion over 5 h after experimental meals containing 0.42 g (A, n = 33) or 1.50 g (H, n = 36) of [15N]-milk protein were given to Wistar rats. The basal value was determined on seven rats. All the rats were previously adapted to an adequate protein diet (14 g per 100 g). Means ± SEM, *: P < 0.05, Meal A vs. Meal H.

View larger version (12K): [in this window] [in a new window]   Figure 2. [15N]-isotopic enrichment (atom % excess) of plasma and urinary urea in Wistar rats given Meal A (n = 33) and H (n = 36) (0.42 or 1.50 g of [15N]-milk protein, respectively). The basal value was determined on seven rats. All the rats were previously adapted to an adequate protein diet (14 g per 100 g). Means ± SEM, *: P < 0.05, Meal A vs. Meal H.

Figure 3 shows concentrations of plasma AA that rose significantly after meal H. There were no significant variations from the basal value (food-deprived group) in plasma AA concentrations in rats fed Meal A. Only certain AA were affected by Meal H, particularly the branched-chain AA (BCAA: valine, leucine and isoleucine) concentrations of which reached a peak 3 h after the meal at 324.4 ± 58.3 µmol/L, 177.2 ± 36.4 µmol/L and 91.9 ± 16.4 µmol/L, respectively. These values were significantly higher than those measured in rats fed Meal A. Similar trends were observed for threonine, phenylalanine and proline, concentrations of which peaked 3 h after the meal (peak concentrations: 388.9 ± 22.7 µmol/L, 61.3 ± 8.1 µmol/L and 298.2 ± 36.2 µmol/L, respectively). In rats fed Meal H, the plasma methionine concentration was significantly increased after 2 h. As for the other plasma AA, there were no significant differences between the two groups, and plasma concentrations remained stable during the 5-h postprandial period.

View larger version (16K): [in this window] [in a new window]   Figure 3. Plasma concentration of several amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine and proline) after Wistar rats were fed a meal containing either 0.42 g (Meal A, n = 33) or 1.50 g (Meal H, n = 36) [15N]-milk protein. The basal value was determined on seven rats. All the rats were previously adapted to an adequate protein diet (14g per 100 g). Means ± SEM *: P < 0.05, Meal A vs. Meal H, °: different from basal value (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 4. [15N]-isotopic enrichment (atom % excess) of plasma lysine and leucine in Wistar rats fed Meal A (n = 33) or Meal H (n = 36) (0.42 and 1.50 g of [15N]-milk protein, respectively). The basal value was determined on seven rats. All the rats were previously adapted to an adequate protein diet (14 g per 100 g). Means ± SEM *: P < 0.05, Meal A vs. Meal H.

The incorporation of [¹⁵N] exogenous nitrogen was studied in the P and NP fractions of the small intestinal and colon mucosa, liver, kidney and muscle. For all tissues, [¹⁵N]-enrichment of the P and NP fractions was significantly higher in rats fed Meal H than in those fed Meal A, with the exception of the P fraction in muscle (Fig. 5 ). In general, [¹⁵N]-enrichment of the NP fraction was higher than that of the P fraction in all tissues, especially in group H. Moreover, the [¹⁵N] kinetics differed in the two N fractions of tissues. [¹⁵N]-enrichment increased steadily in the P fraction of all tissues except muscle during the observation period. In the NP fraction, [¹⁵N]-enrichment kinetics differed as a function of tissue site (splanchnic or peripheral). Splanchnic P fraction enrichment increased more markedly than peripheral tissue enrichment. Enrichment in the P fraction of the gut mucosa varied, depending on the sampling site, and was significantly higher in the proximal part (0.0075 APE in group H) than in the distal part (0.0043 APE in group H).

View larger version (24K): [in this window] [in a new window]   Figure 5. [15N]-isotopic enrichment (atom % excess) of the protein (P) and nonprotein (NP) fractions of tissues in Wistar rats given Meal A (0.42 g of [15N]-milk protein, n = 33) or Meal H (1.50 g of [15N]-milk protein, n = 36). The basal value was determined on seven rats. All the rats were previously adapted to an adequate protein diet (14 g per 100 g). Means ± SEM, *: P < 0.05 Meal A vs. Meal H for the nonprotein (NP) fraction, °: P < 0.05, Meal A vs. Meal H for the protein (P) fraction.

	DISCUSSION

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Exogenous nitrogen in the splanchnic bed.

Splanchnic tissues exhibited the highest [¹⁵N]-enrichments. As expected, enrichment was higher in the proximal small intestinal mucosa, suggesting preferential utilization of the luminal dietary AA for as long as they were available. This was illustrated by the diminishing gradient of both [¹⁵N]-enrichment and dietary nitrogen incorporation in the P fraction along the small intestinal mucosa (Fig. 5 and Table 5 ). This result is supported by Metges et al. (1999a) , who showed that about 40 and 3% of [¹³C]-leucine was incorporated in proximal and distal small intestinal mucosa protein 5 h after a single meal of uniformly [¹³C]-labeled casein (14% protein) had been given to adapted animals.

The increased level of dietary nitrogen incorporated in the three parts of small intestinal mucosa after a meal containing a large quantity of protein highlights the question concerning the capacity of the protein synthesis system to respond rapidly to an acute rise in protein intake. To our knowledge, no study has reported the effects of a single high-protein meal on protein synthesis rates in the intestine. However, a recent work showed no change in the fractional protein synthesis rate in the duodenum of humans after feeding or a 36-h period of fasting (Bouteloup-Demange et al. 1998 ). This suggests that in rats fed Meal A, the capacity for the incorporation of dietary AA into protein synthesis was far from being saturated, and that an additional surplus could enter the anabolic pathways in rats given Meal H. However, we observed that only 3.6% of ingested nitrogen (i.e., 0.155 mmol) was fixed into the small intestinal mucosa protein in group A, and this value fell to 1.8% (i.e., 0.242 mmol) in group H, suggesting saturation of the synthetic capacities.

Using an arterio-venous catheterization technique combined with the infusion of a [¹³C]-tracer, Stoll et al. (1999) reported a 2% incorporation of dietary phenylalanine into mucosal protein. In another study, this value reached 6 to 7% for lysine, threonine and leucine (Stoll et al. 1998a ). These results are similar to our dietary [¹⁵N] recovery in the mucosal protein (Meal A: 3.6%; Meal H: 1.8%), although the interpretation of [¹³C] and [¹⁵N]-tracer data are not precisely the same because of transamination.

The dietary or transformed AA not taken up during the mucosal first pass reach the liver in proportions ranging from 40 to 70% of the ingested load, depending on the AA (Wu 1998 ). The liver NP fraction represents the peptides and free AA that are either released into the plasma, oxidized or incorporated into liver protein. The high enrichment observed in group H may indicate the role of the NP fraction as a buffer or transient pool from which AA are directed into the different pathways.

The incorporation of dietary nitrogen into hepatic protein was 5.7 (i.e., 0.247 mmol) and 4.4% (i.e., 0.596 mmol) of the amount ingested in groups A and H, respectively (Table 5) . These figures are very similar to the values calculated from the results reported by Stoll et al. (1998a) in piglets for threonine and lysine (5 and 8%, respectively) and for leucine and phenylalanine (8%), even if the latter two AA are involved in transamination. The total splanchnic extraction of dietary nitrogen for protein synthesis reached about 12.7% (9% for the constitutive liver protein) in rats fed Meal A, but only 9.8% (6.4% for the constitutive liver protein) in rats fed Meal H. This suggests a rapid saturation of protein synthesis capacities. Because nearly all studies reporting the effect of different protein intakes on protein synthesis focus on low-protein levels (i.e., below requirements), our results are not really comparable with other data. Nevertheless, Eisenstein and Harper (1991) demonstrated in vitro that liver protein synthesis was more sensitive to changes in protein intake in the range 0 to 15% of the total energy ingested, than to changes in doses above 15%. In vivo, Hayase et al. (1998) found a significant increase in the fractional synthesis rate of liver protein when the protein dietary content was raised from 0 to 20%. On the other hand it was shown in humans that the effect of a high dietary protein intake was not so much an increase of the protein synthesis rate but a decrease of the protein breakdown (Garlick et al. 1999 ). Plasma proteins, and especially albumin, which represent 45% of the circulating proteins in the rat (Waynforth 1980 ), incorporated 3.7% (i.e., 0.159 mmol) and 3.4% (i.e., 0.451 mmol) of the amount ingested in group A and group H, respectively. As reported by De Feo et al. (1992) , albumin synthesis is stimulated by feeding and that could prevent irreversible oxidative losses of a large fraction of ingested AA.

The level of protein intake also has a definite effect on the rate of stomach emptying. The quantity of nitrogen recovered after 5 h in rats fed Meal A was dramatically less than that recovered in rats fed Meal H (0.2 and 5.4% of ingested nitrogen, respectively) although in group H, only 3.6 times the amount of protein was ingested when compared with group A. This is consistent with earlier reports by Peraino et al. (1959) showing a deceleration in gastric emptying subsequent to an elevation of dietary protein. This result is of particular importance because it suggests the presence of major regulation at the gastric step, in order to prevent a dramatic increase in dietary nitrogen in the periphery as far as the catabolic capacities of the liver are exceeded. Moreover, it could explain the transient anorexic response induced by a high-protein meal given for the fist time to rats (Peters and Harper 1987 ). This negative feedback on both the stomach emptying rate and food intake could be associated with chemical (AA, catabolic products), biochemical (catabolic/anabolic) and/or physical signals translated by the vagus nerve (Phillips and Powley 1996 and 1998 ). The mechanisms and signals involved in these processes still require clarification.

Splanchnic catabolism and AA availability in the blood.

Our results show that protein synthesis does not appear to be the principal means of regulating postprandial protein metabolism. As reported with a [¹³C]-labeled AA approach (Stoll et al. 1998b ), catabolism plays an important role, since only a small difference in exogenous nitrogen incorporation into proteins occurred between groups when it was compared with the highly differing deamination data. This reflects the fact that most of the surplus dietary AA provided by Meal H entered the catabolic pathways, and only a small amount is disposed of via protein synthesis.

The use of a [¹⁵N] tracer provided interesting information on dietary AA degradation, which could not be obtained with [¹³C] and/or [²H] tracers. In our case, catabolism measured by the postprandial conversion of dietary nitrogen to body and urinary urea (i.e., deamination) indicated the amount of nitrogen of dietary origin which was finally and irreversibly lost. This amount (evaluated 5 h after the meal) was only 7.4% of ingested nitrogen after Meal A and rose to 18.3% after Meal H. These values are consistent with the data obtained in humans who had ingested 30 g of milk protein together with carbohydrates (Gaudichon et al. 1999 ). In rats, the kinetics of postprandial urea [¹⁵N]-labeling differed markedly between animals ingesting Meal A or Meal H (Fig. 2) . In the first case, the enrichment of both plasma and urinary urea rapidly reached a plateau (at 3 h), whereas it increased over a longer period in rats ingesting Meal H. The labeling kinetics of plasma urea, which were still increasing after 5 h in group H, support the idea that postprandial deamination over an extended period would reach higher levels in rats fed Meal H than the total 18.3% reported. This difference is not surprising, since the enzymes responsible for splanchnic catabolism were not stimulated by previous adaptation to a high-protein diet although the time course for optimal activity of hepatic enzymes is highly variable. It has been reported that the activities of some specific enzymes rose markedly after an adaptational period (15–21 d), thereby increasing the capacity for AA catabolism (Colombo et al. 1992 , Moundras et al. 1993 ), and that AA transport in the hepatocytes was stimulated (Fafournoux et al. 1990 , Rémésy et al. 1988 ). Anderson et al. (1968) showed that an adaptation period of at least 3 d to a 50% casein diet is necessary to optimize serine dehydratase activity, whereas some enzymes (such as glutaminase) respond immediately or within 2 h to an acute influx of dietary AA (Ewart and Brosnan 1993 ). As a consequence, plasma threonine concentration increased in rats fed Meal H while no significant changes were observed for any AA in rats fed Meal A (Fig. 3) . BCAA were particularly responsive to Meal H, a result consistent with the findings of Peters and Harper (1987) and Semon et al. (1988) . This marked increase in BCAA plasma concentrations can be explained by the fact that BCAA are poorly metabolized by the liver, due to a low branched-chain amino transferase capacity (Harper et al. 1984 , Torres et al. 1998 ). An interesting outcome is given by the GC-C-IRMS analysis. Indeed, although the amount of ingested AA in H group was 3.5 times that ingested in group A, the amount of [¹⁵N]-labeled AA appearing in the blood was 5 times for leucine and 6 times for valine that measured in group A (Table 4) . This traduces a low oxidation capacity of BCAA after a high-protein meal regarding the surplus that suddenly reaches the liver. In contrast, [¹⁵N]-labeled lysine and threonine appeared 2.6 and 2.9 times that in group A, reflecting the higher capacity of the liver to oxidize those AA. Lastly, the lowest differences between groups were obtained for dispensable AA, especially for alanine and glutamine + glutamate. Since the small intestinal mucosa extracts 98% of luminal glutamate and catabolizes about 70% (Wu 1998 ), exogenous nitrogen is partly transferred to other AA and no longer appears in the plasma glutamate pool, although it is the most abundant AA in milk protein. This could explain why the enrichment of the plasma glutamate pool is not the highest. The case of tyrosine is unique since it is the only dispensable AA that appears under the [¹⁵N]-labeled form {AA concentration x [¹⁵N]-enrichment of AA/[¹⁵N]-enrichment of meal} at a level three times higher in group H as compared to group A. It has been reported that the conversion rate of phenylalanine to tyrosine is high, since it can reach 85% of the phenylalanine extracted by the splanchnic bed (Matthews et al. 1993 ). This could also explain how the plasma tyrosine pool reaches high enrichments. Even if the use of [¹⁵N] does not allow measurement of the fate of dietary AA because of the transaminations resulting in [¹⁵N] exchanges (with the exception of lysine and threonine and possibly also proline and histidine (Metges et al. 1999b ), it provides information on the enrichment dynamics of every plasma AA pool in dietary nitrogen and is useful for qualitative comparisons of different nutritional conditions.

Incorporation of exogenous nitrogen in peripheral tissues.

The [¹⁵N]-enrichment kinetics observed in P and NP fractions of peripheral tissues differed from those observed in splanchnic organs. Indeed, the colon mucosa (which can be considered as a peripheral organ), kidney and especially muscle exhibited very low [¹⁵N]-enrichments (Fig. 5) . This was due both to a lower availability of [¹⁵N] in the periphery than in the splanchnic area and to a lower protein synthesis rate. The low fractional synthetic rate (about 1 to 15%/d) of skeletal muscle protein is well-documented (Garlick et al. 1973 and 1994 , Laurent et al. 1984 , McNurlan and Garlick 1989 ) and explains why muscle protein enrichment was hardly increased and was not sensitive to the protein content of the meal within the experimental period. Nevertheless, given the major contribution of the muscle to whole body protein, dietary nitrogen was mainly sequestered in muscle (Table 5) . Few available data concern the effects of various protein intakes on muscle synthesis capacities. In a dietary adaptation study (14 d of 2 to 40% casein diets), Laurent et al. (1984) found no difference in muscle protein synthesis rates with intakes of 6, 20 or 40% dietary casein. With reference to our observations in splanchnic tissues, it could be argued that protein synthesis is not stimulated by the acute ingestion of a high-protein meal but that the higher availability of dietary nitrogen, although markedly buffered by the splanchnic first pass, led to a higher appearance of dietary nitrogen in muscle. The results in the kidney are remarkable: almost no dietary nitrogen was fixed in the P fraction, and this was the only organ which accumulated more dietary nitrogen in the NP than in the P fraction (Table 5) . However, a relatively high protein synthesis rate of about 40–50%/d has been reported in the kidneys (Hayase et al. 1998 , Tessari et al. 1996 ). One hypothesis is that the exchange rate of free AA between the extracellular and intracellular pools is particularly low, as was calculated by Carraro et al. (1991) in their 16-pool model for leucine kinetics in dogs. A more likely explanation, however, is that the NP fraction was very rich in urea, ammonia and glutamine, especially in group H. The colon mucosa contained only small amounts of dietary nitrogen (0.1 that in the duodenum mucosa) which is in accordance with the low synthesis rate reported of about 10%/d (Garlick et al. 1994 , Rennie et al. 1994 ) (Table 5) .

Finally, the postprandial distribution of dietary nitrogen shows that 5 h after the meal, a high proportion was retained in muscle despite a low nitrogen incorporation rate. This represents 45% of ingested nitrogen in group A. In contrast, this value was considerably lower in group H (22%) due to: (i) a lower availability of dietary nitrogen for the peripheral tissues and (ii) limited protein synthesis, reflecting the fact that protein synthesis systems were not yet stimulated. This last phenomenon was also observed in the splanchnic area, which incorporated 10.3 and 7.3% of ingested nitrogen in groups A and H, respectively. Under those conditions, 70.8% of ingested nitrogen was recovered in all the samples collected in rats fed Meal A containing 0.42 g of protein, whereas this value was 62.2% in rats fed Meal H containing 1.50 g of protein. A major proportion of the surplus dietary nitrogen was eliminated through urea. This indicates that the efficiency of retention was lower in group H even though the absolute quantity of incorporated dietary nitrogen was higher when compared to group A.

Measuring for the first time the dynamic processes involved in the transfer of dietary nitrogen in the splanchnic and peripheral tissues as well as in almost all plasma AA, this study shows original data and highlights the mechanisms involved in regulating protein metabolism after an acute elevation of protein intake. First, both splanchnic and peripheral tissues incorporated more dietary nitrogen, but this phenomenon was limited by saturation of the protein synthesis capacities. Second, the principal metabolic response was the activation of splanchnic AA catabolism, leading to an increase in urea production. Third, rapidly attained high [¹⁵N]-enrichments were observed for all plasma AA, but due to increased catabolic and anabolic utilization, most AA plasma concentrations remained constant, and only a few plasma AA, BCAA, threonine, methionine, proline and tyrosine, were sensitive to the greater protein intake. Our [¹⁵N] results strongly suggest a higher catabolism of all the dispensable AA and some indispensable AA such as lysine and threonine. This study shows that, when a high- protein meal is ingested for the first time, in spite of their capacities to respond to variations in protein ingestion, the body catabolic and anabolic systems are not able to maintain concentrations in the blood and tissues at constant physiological levels for all AA. This indicates that the metabolic systems are limited in their response to AA given in excess, compared with an adequate intake. One of the major regulations identified by this study, in response to the saturation of catabolic capacities, is the deceleration of gastric emptying. This important finding can also be related to the anorexia induced by a high- protein meal reported in the literature. These two mechanisms may constitute the critical responses to an acute elevation of dietary protein.

	ACKNOWLEDGMENTS

 
We are indebted to Christiane Larue-Achagiotis for her helpful scientific comments and contribution to surgical procedures. Sophie Daré and Catherine Luengo are gratefully thanked for their technical assistance.

	FOOTNOTES

 
² Abbreviations used: AA, amino acid; AP, atom %; APE, atom % excess; BCAA, branched-chain amino acids; BW, body weight; GC, gas chromatograph; GC-C-IRMS, gas chromatography–combustion–isotopic ratio mass spectrometry; Meal A, meal containing 0.42 g of protein; Meal H, meal containing 1.50 g of protein; NP fraction, nonprotein fraction; NPP, privaloyl-I-propyl; P fraction, protein fraction; TCA, trichloroacetic acid.

Manuscript received February 21, 2000. Initial review completed March 13, 2000. Revision accepted May 15, 2000.

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