INTRODUCTION

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AMINO ACID TRANSPORT in the apical membrane of intestinal absorptive cells is regulated, among other factors, by dietary substrate levels, although the pattern of regulation may differ according to the role in metabolism of the transported substrate (6). For nonessential nutrients, such as some amino acids, the adaptive regulation hypothesis (6) predicts that an increase in intake of the nutrient upregulates its own transport, whereas for nutrients yielding calories but showing some toxicity, such as some essential amino acids, the expected pattern of regulation is also an upregulation but of smaller magnitude, which reduces the risk of intoxication.

In a previous study on the chicken intestine (14) we showed that dietary L-lysine supplementation results in upregulation of L-lysine transport via the b^0,+ and y⁺m systems. In rats supplemented with L-phenylalanine and L-tyrosine, Wapnir et al. (16) observed a reduction in the intestinal transport of L-phenylalanine and L-tyrosine, suggesting a pattern of downregulation rather than upregulation. Because L-phenylalanine and L-tyrosine show higher toxicity than L-lysine (8), these results indicate that the toxicity of the substrate may also determine differences in the pattern of regulation.

L-Methionine is an essential amino acid in poultry nutrition, showing the highest toxicity (8). L-Methionine is transported in the apical membrane of the chicken jejunum by multiple pathways: systems b^0,+-like and y⁺m, shared with cationic amino acids and also by systems L- and B-like, specific for neutral amino acids (12) (see Table 1). Here we have examined the effect of dietary methionine supplementation on L-methionine transport and have tested the adaptive hypothesis for an amino acid with higher toxicity than L-lysine. We have also assessed the extent to which the supplemented diet affects L-lysine uptake via the transport systems shared with L-methionine. The results indicate that dietary supplementation with L-methionine causes a reduction in the uptake of L-methionine and L-lysine, mainly due to a decrease in transport capacity of systems y⁺m, L- and B-like.

	METHODS

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Animals and diets. Male Label chickens (Gallus gallus domesticus L.) were fed ad libitum from the day of hatch to the first week of life with a standard diet (control diet) and then randomly divided into two groups and fed for 5 additional weeks with either the same diet, containing 25 g methionine/kg dietary protein (6 g DL-methionine/kg protein plus 19 g/kg protein of added L-methionine) or a diet enriched with L-methionine (Met diet) to reach a value of 45 g/kg dietary protein (9 g/kg diet). Food consumption and body weight were measured twice a week throughout the experiment. Diets were isoenergetic (13 MJ/kg food) and formulated to contain 20% protein, 5.52% lipid, and 38.5% carbohydrate. The composition of the control diet in essential amino acids was (in g/kg diet) 5 DL-methionine, 9.8 L-isoleucine, 23 L-leucine, 8 L-threonine, 13.2 L-arginine, 2.1 L-tryptophan, 9.6 L-lysine, and 9.1 DL-methionine-L-cysteine. The formulation, preparation, and chemical analyses of diets were performed by IRTA-Mas Bové (Reus, Spain). Animals (6-wk-old chickens) were killed in the morning by neck fracture, without previous starvation. The jejunum (from the end of the duodenal loop to the Meckel diverticle) was removed, immediately flushed with ice-cold saline, opened lengthwise, frozen in liquid N₂, and then stored at 80°C. Manipulation and experimental procedures are in accordance with the Spanish regulations for the use and handling of experimental animals.

Isolation of brush-border membrane vesicles. Brush-border membrane vesicles (BBMV) were prepared using the Mg²⁺-precipitation method of Kessler et al. (9) as previously described (13). The composition of the intravesicular medium was 300 mmol/l mannitol, 0.1 mmol/l MgSO₄ · 7H₂O, 0.02% LiN₃, and 20 mmol/l HEPES-Tris, pH 7.4. Vesicles were diluted to a final protein concentration of 20-30 mg/ml, frozen, and stored in liquid N₂ in 150 µl aliquots for no more than 5 mo. Each isolation batch corresponds to the jejunum of one chicken, and in RESULTS n indicates the number of chickens or membrane preparations.

Protein and enzyme assays. The purity of the membrane preparations was routinely assayed from sucrase and ATPase activities, according to Dahlqvist (1) and del Castillo and Robinson (2), respectively. Protein was determined using the Bio-Rad protein assay, with bovine serum albumin as standard.

Uptake assays. Transport experiments were carried out at 37°C for incubation periods ranging from 1 s to 1 h using the rapid filtration technique previously described (13). The vesicles were incubated under isotonic conditions (320 mosm/kg) in an incubation medium containing 100 mmol/l mannitol, 0.2 mmol/l MgSO₄ · 7H₂O, 0.02% LiN₃, 20 mmol/l HEPES-Tris (pH 7.4), 100 mmol/l of NaSCN or KSCN, and the appropriate labeled and unlabeled D-glucose, L-methionine, or L-lysine concentration. Incubation was stopped by the addition of 2 ml ice-cold stop solution (150 mmol/l KSCN, 0.02% LiN₃, and 20 mmol/l HEPES-Tris, pH 7.4). Samples were rapidly filtered under negative pressure through prewetted and chilled 0.22 µm cellulose acetate/nitrate filters (Millipore GSWP02500). The filters were rinsed four times with 2 ml ice-cold stop solution and dissolved in Biogreen-6 cocktail from Sharlau (Barcelona, Spain). Radioactivity was determined by liquid scintillation counting (Packard Tri-Carb, model 1500). Nonspecific tracer fixation to the filters was obtained by adding ice-cold stopping solution to reaction tubes immediately before addition of the vesicles. Experiments were performed with at least three different membrane preparations, each in triplicate.

Chemicals. All unlabeled reagents, including the L-methionine used for diet enrichment, were from Sigma Chemical (St. Louis, MO). D-[U-¹⁴C]glucose, L-[methyl-³H]- methionine, L-[methyl-¹⁴C]methionine, and L-[4,5-³H(N)]lysine were obtained from New England Nuclear Research Products (Dreieich, Germany).

Kinetic study. The kinetic characterization of L-methionine and L-lysine transport was performed from self-inhibition curves using 0.5 µmol/l L-[³H]methionine, 50 µmol/l L-[¹⁴C]- methionine, 10 µmol/l L-[¹⁴C]lysine, and 0.25 µmol/l L-[³H]lysine as substrate and varying concentrations of unlabeled amino acids on the cis side. The incubation time tested was 2 s for L-methionine and 3 s for L-lysine, because, for these substrate concentrations, transport was linear in both dietary treatments for 3 and for 5 s, respectively (data not shown). The nonsaturable component was measured in the presence of high unlabeled amino acid concentrations (30 and 20 mmol/l for L-methionine and L-lysine, respectively). This component was then subtracted from total amino acid influx. Diffusion constant (K_D) values calculated for both substrates show no dietary modifications (P  0.05, see Tables 5, 7, and 8).

The rates of mediated transport were fitted by nonlinear regression analysis from plots generated by the Biosoft Enzfitter statistical package (Cambridge, UK). The best fit was assigned, according to the criteria of Motulsky and Ransnas (10), to the fit showing the lowest as well as significantly different residual sums of squares (P < 0.05).

Transport equations. The kinetic parameters were calculated from the relative transport rates v/v₀ (labeled substrate uptake in the presence and absence of unlabeled amino acid) following the strategy described by Devés et al. (3) as previously described (12). In the presence of very low substrate concentration, the transport through one (a) or two transport systems (a and b) can be described by the following equations

(1)

(2)

where K_ia and K_ib are the inhibition constants of systems a and b, respectively, [I] is the concentration of unlabeled inhibitor, and F_a/b is the "permeability" ratio, which indicates the relative contribution of each system to the total influx.

(3)

where K_ma and K_mb and V_maxa and V_maxb are, respectively, the affinity and the maximum capacity constants of systems a and b.

When substrate concentration was 10 or 50 µmol/l, the following equations, for one (equation 4) or two (equation 5) transport systems were used

(4)

(5)

where [S] is the substrate concentration.

The experimental strategy for the kinetic analysis consisted of the following steps. 1) The permeability ratio (F_a/b) and K_m were calculated from L-methionine or L-lysine self-inhibition relative transport rates (v/v₀). Because L-methionine transport in this epithelium has been described to be mediated at least by four transport systems (12), self-inhibition experiments were performed in the presence of 10 mmol/l L-lysine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) to reduce the number of functional pathways, thus simplifying the kinetic analysis. 2) V_max values were calculated from self-inhibition transport rates (v), taking the K_m calculated in step 1 as fixed values. To facilitate comparison of the kinetic constants, the V_max values for L-methionine and L-lysine were normalized to 1 s incubation. 3) K_i was calculated from relative transport rates in the presence of increasing concentrations of L-lysine or BCH for L-methionine transport and L-methionine for L-lysine uptake, taking the K_m values calculated in step 1 as fixed.

Data analysis. The data were compared using ANOVA and Snedecor's F test. Kinetic constants were compared by Student's t-test. In all cases, a P < 0.05 value was considered statistically significant.

	RESULTS

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Effect of diets on body weight. Chickens fed Met diet for 5 wk showed no differences in body weight (control diet, 1,140 ± 20; Met diet, 1,137 ± 18 g, means ± SE, n = 40) or in the efficiency of food utilization (control, 0.32 ± 0.02; Met, 0.33 ± 0.03 g wt gain/g food intake).

Properties of BBMV. The purity of the vesicles isolated from both control and Met diet-fed animals was checked from sucrase and Na⁺-K⁺-ATPase enrichment factors. The results (Table 2) show low basolateral membrane contamination for both diets (P  0.05). The ability of the vesicles to transport D-glucose was taken as an index of the BBMV functionality. D-Glucose uptake (100 µmol/l) was determined under inwardly directed NaSCN or KSCN gradients (initial intravesicular salt concentration equals 0 mmol/l and extravesicular salt concentration equals 100 mmol/l). Table 2 shows that vesicles behave as expected and that no differences were observed between diets.

Time course of L-[¹⁴C]methionine. Figure 1 shows time-dependent uptake of L-methionine (100 µmol/l) under zero-trans 100 mmol/l NaSCN or KSCN gradient. In the presence of Na⁺ (Fig. 1A), there was a transient overshoot that peaked at 2 s incubation and was higher in control than in vesicles from Met-fed animals. Moreover, a slight dietary reduction was observed when L-methionine uptake was measured in the presence of K⁺, whereas no effects were detected in the presence of K⁺ plus 50 mmol/l unlabeled L-methionine (Fig. 1B). In these conditions, no overshoot was observed, and initial uptake in the presence of unlabeled amino acid was slower for both diets, suggesting a similar nonmediated component. The equilibrium values (60 min) were neither affected by the diet nor by the incubation conditions, indicating no modifications in either vesicular volume or L-methionine binding to the membrane.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Time course for uptake of 100 µmol/l L-methionine under zero-trans. A: NaSCN [, control diet; , L-methionine-enriched (Met) diet]; B: KSCN (, , control and Met diet, respectively) or KSCN plus 50 mmol/l unlabeled L-methionine (, , control and Met diet, respectively). Membranes were prepared in 300 mmol/l mannitol, 0.1 mmol/l MgSO4 · 7H2O, 0.02% LiN3, and 20 mmol/l HEPES-Tris, pH 7.4. Incubation medium contained 100 mmol/l salt, 100 mmol/l mannitol, 0.1 mmol/l MgSO4 · 7H2O, 0.02% LiN3, 20 mmol/l HEPES-Tris, pH 7.4, and 100 µmol/l L-methionine (50 µmol/l L-[14C]methionine). Osmolarity was maintained by reducing mannitol concentration. Values represent means ± SE of 4 or 5 membrane preparations. Only SE that exceed size of symbol are shown.

Cis-inhibition experiments. Cis-inhibition experiments were performed to characterize the transport components involved in L-methionine influx in Met vesicles. The effect of relatively high concentrations of unlabeled amino acids on L-methionine influx (2 s, 50 µmol/l), both in the absence and in the presence of 0.5 mmol/l N-ethylmaleimide (NEM), was studied under KSCN gradient (Table 3). The results indicate the presence of two components showing different sensitivity to L-lysine and BCH (see Table 1). The L-lysine-sensitive (BCH resistant) component was partially inhibited by NEM, and the remaining NEM-insensitive component was inhibited by L-cystine. In addition, no differences between L-methionine influx in the presence of 10 mmol/l unlabeled L-methionine in control and Met-fed animals were observed (data not shown).

Under a Na⁺ gradient (Table 4), Met vesicles show no differences (P  0.05) in L-methionine influx in the presence of high unlabeled L-methionine concentration compared with K⁺ conditions. Therefore, cis-inhibition fluxes of the Na⁺-dependent component were calculated by subtracting KSCN from NaSCN values. The results show that the Na⁺-dependent L-methionine influx is not affected by L-lysine and that BCH exerts a small but significant inhibition on L-methionine uptake.

Kinetic analysis of L-methionine transport. Taking into account the transport components detected in cis-inhibition experiments for L-methionine transport, the kinetic analysis was done in the presence of 10 mmol/l BCH or L-lysine under zero-trans KSCN or NaSCN gradients (Figs. 2 and 3, respectively). In control animals, K_m values calculated were in the micromolar range for system b^0,+-like and in the millimolar range for systems y⁺m and L-like (12). Therefore, substrate concentrations used were 0.5 µmol/l L-[³H]methionine or 50 µmol/l L-[¹⁴C]methionine, with BCH and L-lysine present in the incubation media, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   L-Methionine self-inhibition experiments under zero-trans 100 mmol/l KSCN gradient. A: total L-[3H]methionine (0.5 µmol/l) influx in presence of increasing concentrations of unlabeled L-methionine and 10 mmol/l 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH; , control diet; , Met diet). B: total L-[14C]methionine (50 µmol/l) influx in presence of increasing concentrations of unlabeled L-methionine and 10 mmol/l L-lysine (, control diet; , Met diet). Vesicles were prepared and incubated as described in Fig. 1. Values represent means ± SE of 4 or 5 membrane preparations. Only SE that exceed size of symbol are shown.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   L-Methionine self-inhibition experiments under zero-trans 100 mmol/l NaSCN gradient. A: total L-[3H]methionine (0.5 µmol/l) influx in presence of increasing concentrations of unlabeled L-methionine and 10 mmol/l BCH (, control diet; , Met diet). B: total L-[14C]methionine (50 µmol/l) influx across Na+-dependent component in presence of increasing concentrations of unlabeled L-methionine and 10 mmol/l L-lysine (, control diet; , Met diet), calculated by subtracting values obtained in absence of Na+ (Fig. 2B) from those obtained under Na+ gradient (data not shown). Vesicles were prepared and incubated as described in Fig. 1. Values represent means ± SE of 4 or 5 membrane preparations. Only SE that exceed size of symbol are shown.

In the presence of K⁺, the best fit for the BCH-resistant component in Met-fed chickens was obtained by considering a model including two transport systems (Fig. 2, Table 5): a high-affinity low-capacity transport system (K_m = 16 ± 3.0 µmol/l and V_max = 1.45 ± 0.07 pmol · mg protein¹ · s¹) and a low-affinity, high-capacity transport mechanism (K_m = 2.4 ± 0.18 mmol/l and V_max = 173 ± 1.5 pmol · mg protein¹ · s¹). The kinetic characteristics of these pathways are consistent with those of the transport systems described in control animals and identified with systems b^0,+ (high affinity, low capacity) and y⁺m (low affinity, high capacity; Ref. 12). The results show no dietary effect on the b^0,+-like system and a significant 30% reduction in y⁺m V_max without any effect on the K_m values.

Results obtained in the presence of 10 mmol/l L-lysine show the best fit when a single transport system, identified with system L in control animals (12), is considered. In Met-fed chickens, this transport system shows no changes in the affinity constant and a 30% reduction in V_max values.

The inhibition constants for L-lysine and BCH (Table 6) calculated from the inhibition curves indicate no dietary effect for the three transport systems considered (data not shown).

When K⁺ was replaced by Na⁺, the BCH-resistant component showed the best fit for two transport systems (Fig. 3, Table 7); the first one is system b^0,+-like, which shows similar kinetic parameters when compared with K⁺ results and no dietary modifications (control: K_m = 8.0 ± 2.0 µmol/l and V_max = 1.5 ± 0.03 pmol · mg protein¹ · s¹; Met: K_m = 10 ± 3.0 µmol/l and V_max = 1.35 ± 0.21 pmol · mg protein¹ · s¹), and the second one is system y⁺m, which shows a similar dietary effect as in K⁺ conditions (26% reduction in V_max).

The kinetic constants of the Na⁺-dependent component, identified with system B, were calculated following the strategy described by Soriano-García et al. (12). L-Methionine self-inhibition fluxes in the presence of 10 mmol/l L-lysine under K⁺ gradient were subtracted from the values obtained in the presence of Na⁺. The results (Table 7) show the best fit for a single transport system with a 30% decrease in K_m and a 51% decrease in V_max due to dietary adaptation.

Kinetic analysis of L-lysine uptake. L-[³H]lysine (0.25 µmol/l) self-inhibition experiments were done in the presence of zero-trans KSCN or NaSCN gradients (Fig. 4, Table 8). The results confirm the presence of the transport systems b^0,+-like and y⁺m. In the presence of K⁺ and Na⁺, b^0,+-like system showed no dietary modifications in the kinetic parameters, whereas a decrease in y⁺m capacity was observed in Met-fed animals (a 19% reduction in the presence of KSCN and a 28% reduction with NaSCN). Moreover, the Na⁺ sensitivity of this transport system previously detected in control animals (V_max increase, K⁺ vs. Na⁺) was reduced by L-methionine supplementation.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Total L-[3H]lysine (0.25 µmol/l) influx in presence of varying concentrations of unlabeled L-lysine (, control diet; , Met diet) under zero-trans 100 mmol/l NaSCN (A) or KSCN (B) gradient. Vesicles were prepared and incubated as described in Fig. 1. Values represent means ± SE of 4 or 5 membrane preparations. Only SE that exceed size of symbol are shown. Insets: magnification of 0.0-0.01 L-lysine concentration range.

The inhibition constants for L-methionine on L-lysine influx (Table 6), calculated from the inhibition curves in the presence of KSCN, showed no dietary modifications (data not shown).

	DISCUSSION

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In a previous study we reported that the transport of L-methionine in the chicken jejunum was mediated by multiple transport systems, two of which were shared with cationic amino acids (12). The results of the present study, from both cis-inhibition and kinetic experiments, indicate that the same transport pathways are present in the animals fed the Met diet, that is, systems y⁺m and b^0,+-like for neutral and cationic amino acids and systems L- and B-like specific for neutral amino acids.

Dietary effects on kinetic constants. The kinetic results of Met-fed chickens indicate that L-methionine-enrichment exerts a similar effect on the uptake of both L-methionine and L-lysine. Substrate influx through system b^0,+-like is not affected by the dietary treatment either in the presence or in the absence of Na⁺. In contrast, system y⁺m shows a downregulation profile due to a significant reduction in V_max, without any effect on the K_m. For L-methionine, this reduction is similar in the presence and in the absence of Na⁺ (26 and 30%, respectively), whereas for L-lysine the reduction is more pronounced under Na⁺ gradient (KSCN, 19%; NaSCN, 28%).

System y⁺m shows higher affinity and capacity for L-methionine in the presence of Na⁺ (12) and similar affinity and higher capacity for L-lysine (13). The differences between Na⁺ and K⁺ results have been attributed to a membrane potential effect (13); in the case of L-methionine, the sensitivity to membrane potential may be due to the charge associated with the protein carrier (4), whereas for L-lysine, the cationic nature of this amino acid might also contribute to the response of the transporter to the presence of Na⁺. In Met-fed chickens, the differences detected when K⁺ was replaced by Na⁺ are maintained for L-methionine and reduced for L-lysine. These results probably reflect a different sensitivity of system y⁺m to Na⁺, depending on the nature of the substrate transported as previously reported (14).

The transport systems specific for neutral amino acids, L- and B-like, show a downregulation profile similar to that described for system y⁺m (30 and 51% decrease in V_max, respectively). However, the K_m for the B-like system is reduced 30% in Met-fed chickens. Changes in carrier affinity have been observed during ontogenic development and enterocyte maturation (5) and have rarely been attributed to dietary regulation (17). In physiological conditions, the concentration of individual amino acids in the lumen ranges from 1 to 25 mmol/l (15) and so the regulation of amino acid uptake by mechanisms involving changes in the V_max have a greater effect than regulatory patterns based on changes in the K_m. Therefore, because the contribution of the transport systems to total mediated influx is mainly determined by their V_max, the downregulation effect observed on the high-capacity transport systems may result in a significant reduction of total amino acid transport capacity by the small intestine.

Adaptive regulation. When dietary amino acid levels are increased, the adaptive regulation hypothesis predicts an upregulation of amino acid transport (6). Ferraris and Diamond (6) showed that the regulatory pattern of nutrient absorption differs according to its metabolic and transport properties. Thus essential, toxic amino acids show upregulation but less than nonessential, nontoxic nutrients. However, the results of the present study indicate that supplementation with the essential amino acid L-methionine induces a net reduction in its own uptake, i.e., downregulation, rather than a change in an upregulatory pattern. This can be interpreted as a further "protective" response, because L-methionine shows the highest toxicity among essential amino acids (8). The study of Wapnir et al. (16) also showed reduced amino acid uptake in rats as a result of diet enrichment with L-phenylalanine.

To assess the effect of this adaptive regulation on intestinal amino acid absorption, the contribution of passive diffusion should also be considered. Because K_D values are not reduced by dietary treatment and considering that the concentration of L-methionine would be higher in the intestinal lumen of Met-fed animals, L-methionine uptake via the nonmediated component would be increased in treated chickens. Therefore, the role of the downregulation of the mediated pathways would be essential to reduce the increase in total substrate influx, thus avoiding the toxic effects described for L-methionine excess (8). The intestine would thus regulate amino acid absorption without affecting the substrate uptake across unspecific passive mechanisms that spend less energy than mediated processes.

Dietary effects on animal growth. The effect of Met diet on animal body weight and food consumption was also examined. Dietary methionine supplementation has been described to improve animal growth in chickens fed a diet containing low levels of sulfur amino acids (7). Moreover, Harper et al. (8) found that high methionine levels produce a decrease in body weight, attributed to a reduction in food intake, which is related to amino acid toxicity. In the present study, L-methionine was supplemented in a diet formulated to meet the recommendations of the National Research Council (11) for chicken performance. The results show that the addition of 0.4% L-methionine to a diet containing 0.5% DL-methionine and 20% protein does not affect body growth or efficiency of food utilization. This lack of effect may be related to adequate protein intake, which results in an increase in the capacity of the animal to tolerate disproportionate amounts of amino acids (8, 11), and to downregulation of amino acid transport, which will eventually avoid the adverse effects of L-methionine excess on the growth rate.

Perspectives

Animals fed an excess of dietary methionine show an impairment in body weight gain (8). This effect may reflect nonspecific changes in amino acid transport and in the absorption of other nutrients. In this situation, nonspecific regulatory mechanisms would mask any specific adaptation of the mediated transport. It would also be interesting to examine to what extent this specific adaptive regulation response "protects" the animals from modifications in nutritional parameters.