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Developmental study of alpha-methyl-D-glucoside and L-proline uptake in the small intestine of the White Leghorn chicken
Developmental Study of a-Methyl-D-Glucoside and L-Proline Uptake in the Small Intestine of the White Leghorn Chicken1 M. ESTRELLA SORIANO and JOANA M. PLANAS2 Grup de Transport Intestinal, Departament de Fisiologia-Divisio´ IV, Facultat de Farma`cia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain undergoes substantial changes with age and region studied. Intracellular accumulation of a-methyl-Dglucoside and L-proline was greater in newly hatched chicks and then declined with age in the three regions of the small intestine, except for L-proline transport in ileum, which remained constant during the period studied. The transport mechanisms for each nutrient followed separate developmental patterns along the small intestine during the period studied.
(Key words: White Leghorn, intestinal transport, monosaccharides, development, amino acid) 1998 Poultry Science 77:1347–1353
INTRODUCTION From hatch, the chick intestinal tract undergoes major changes to enable it to extract nutrients from an external solid diet and to absorb them. During this period, there are adaptative changes in gastrointestinal morphology and function that contribute to meeting the increasing metabolic demands during early development (Buddington and Diamond, 1989). Most of the ontogenetic studies on intestinal transport have shown a close relationship between the diet and the capacity to transport nutrients (Ferraris, 1994). However, in most of these studies, the composition of the diet was not kept constant and it is not possible to conclude that the observed changes are due exclusively to feed intake. The process of intestinal development involves such a large number of variables that it is difficult to find the primary cause of age-related changes. In the present study, Gallus gallus domesticus was selected as a useful model for ontogenetic studies
Received for publication October 20, 1997. Accepted for publication May 7, 1998. 1This work was subsidized by grants PB88/0219 and PB91/0159 from Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, Ministerio de Educacio´n y Ciencia, Spain. 2To whom correspondence should be addressed: [email protected]
on intestinal transport because this species can be raised on a diet of constant composition. Studies on the transport of sugars and amino acids in the intestine of chicken during development have been carried out, but the results are contradictory because the separate contributions of multiple mediated pathways were not evaluated, or well-defined regions of the gut were not used (Bogner and Haines, 1964; Holdsworth and Wilson, 1967; Lerner et al., 1976; Raheja et al., 1977; Shehata et al., 1981; Planas et al., 1982 and 1986). In two previous papers (Esteban et al., 1991; Moreno et al., 1996), we have established the developmental patterns and kinetic constants of transport of a-methyl-Dglucoside (aGlc1Me) in the duodenum, jejunum, and ileum of chick during the perinatal period. The important regional and developmental changes found in these studies induced us to extend them until adulthood using not only a monosaccharide but also an amino acid. The purpose of the present study was to determine the capacity to accumulate aGlc1Me and L-Pro as well as to establish the developmental patterns of the mediated and nonmediated component in the different regions of the small intestine of the chicken from the posthatch period until adulthood.
Abbreviation Key: aGlc1Me = a-methyl-D-glucoside; Glc3Me; 3-oxy-methyl-D-glucose; PEG = polyethylene glycol.
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ABSTRACT Development changes in a-methyl-Dglucoside and L-proline accumulation were studied using everted sleeves from the duodenum, jejunum, and ileum of chickens. Six age groups of chickens were used: 1 d and 1, 2, 3, 5 to 6, and 12 to 14 wk. Our results showed the presence of a Na+-dependent mechanism of sugar and amino acid uptake that is already fully developed at hatch. The intestinal transport activity
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SORIANO AND PLANAS TABLE 1. Composition of the diet
Ingredient composition
30.87 26.00 1.50 2.00 3.00 25.80 7.00 0.33 0.05 0.30 2.50 0.50 0.01 0.10 0.04 3,014.00 21.40 0.70 0.35 1.10 0.80 0.25 0.80 0.40
1Mineral premix provides per kilogram of diet: Mn, 50 mg; Zn, 50 mg; Fe, 40 mg; Cu, 5 mg; Se, 0.15 mg. 2Vitamin premix provides per kilogram of diet: vitamin A (as retinyl acetate), 11,250 IU; vitamin E (as dl-a-tocopherol acetate), 32 IU; cholecalciferol, 1,800 IU; thiamine, 5.5 mg; riboflavin, 6.7 mg; pyridoxine, 9.0 mg; cobalamine, 0.01 mg; pantothenic acid, 13.5 mg; niacin, 64.5 mg; folic acid, 1.5 mg; biotin, 0.2 mg.
MATERIAL AND METHODS
Animals Male White Leghorn chickens were used. Animals were obtained from the commercial farm Cooperativa Comarcal d’Avicultura de Reus3 on the day of hatch and maintained under conditions of stable humidity and temperature with a 12:12 h light:dark cycle. The birds had free access to water and feed (Table 1). Experiments were carried out using animals at the following ages: 1 d and 1, 2, 3, 5 to 6, and 12 to 14 wk.
Tissue Uptake of aGlc1Me and L-Pro Birds were killed by decapitation and intestinal segments were removed as follows: duodenal samples were taken from the duodenal loop, jejunal samples were sliced at the level of the yolk stalk, and ileal samples were taken from the end of ileum where the region was connected with mesentery to the ceca. Intestinal segments were trimmed from adherent mesenteric tissue, washed
3Cooperativa Comarcal d’Avicultura, 43280 Reus, Spain. 4NEN Life Science Products, 100 Rue de la Fuse ´ e, B-1130 Brussels,
Belgium. 5Sigma Chemical Co., St. Louis, MO 63178-9916.
Expression of Uptakes Total uptake of aGlc1Me or L-Pro was expressed as nanomoles per 100 mg of tissue and the phloridzinsensitive component of the transport as micromoles per liter of intracellular water.
Materials Labeled compounds [14C(U)]-aGlc1Me, [14C(U)]-L-Pro, and [1,2-3H]-PEG-4000 were purchased from NEN Life Science Products.4 The specific activity and the final concentration of 14C-aGlc1Me were 265 mCi/mmol and 0.04 mCi/mL, respectively. The specific activity and the final concentration of 14C-L-Pro were 290 mCi/mmol and 0.05 mCi/mL, respectively. The specific activity and the final concentration of 3H-PEG-4000 were 8 mCi/mmol and 0.08 mCi/mL, respectively. Unlabeled compounds were obtained from Sigma.5
Statistical Analyses The comparative study of total uptake was performed by fitting the results obtained for each age group and each segment to a horizontal parabola, which was then linearized. The slopes of the resulting lines were compared by ANOVA. Snedecor’s F test was used to detect differences between age groups and intestinal regions (Zivin and Bartko, 1976). A probability level of 5% (P < 0.05) was considered significant.
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Ground yellow corn Wheat Corn gluten feed Corn DDG with solubles Wheat shorts Soybean meal 44 Meat and bone meal Calcium carbonate Dicalcium phosphate Sodium chloride Animal fat Beet molasses L-Lysine·HCl Mineral premix1 Vitamin premix2 Calculated nutrient composition ME, kcal/kg Crude protein, % TSAA, % Met, % Lys, % Thr, % Trp, % Calcium, % Nonphytate phosphorus, %
Percentage
with ice-cold (4 C) saline solution, everted, and cut into segments of about 1 cm. Slices were placed in 10 mL of incubation medium containing (in millimoles per liter): NaCl, 118.5; KCl, 6.2; CaCl 2 , 2.5; KH 2 PO 4 , 1.2; MgSO4·H2O, 1.2; NaHCO3, 24.9, and aGlc1Me or L-Pro, 0.1. The solution was maintained at 37 C, pH 7.4 and bubbled with 95% O2-5% CO2 with adequate stirring. Trace concentrations of 14C-aGlc1Me and 14C-L-Pro were added to quantify the amount of nutrient that was accumulated in the tissue. The passive component for aGlc1Me was determined by adding phloridzin 0.2 mmol/L. For L-Pro, this component was measured by replacing the NaCl with choline chloride in the incubation solution and NaHCO3 with KHCO3. In order to determine the extracellular space, tissue slices were incubated with 3H-polyethylene glycol (PEG)-4000. To calculate the total water content, the intestinal segments were dried at 60 C for 24 h, and the difference between dry weight and total wet weight was determined. Intracellular water of each intestinal region was then estimated by subtracting the extracellular space from the corresponding total water. Tissues were exposed to aGlc1Me or L-Pro at the concentration of 0.1 mmol/L during different times (6, 15, 25, 35, and 45 min). After incubation, the tissues were removed, briefly washed in ice-cold saline, blotted on filter paper, and weighed. Both aGlc1Me and L-Pro were extracted from the tissue using HNO3 0.1 mmol/L overnight at 4 C. Radioactivity in the extract was determined using standard liquid scintillation procedures.
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RESULTS
Total uptake of aGlc1Me Figure 1 shows the total uptake of 0.1 mmol of aGlc1Me/L by pieces of duodenum, jejunum, and ileum at different stages of development during 45 min of incubation. Total uptake of aGlc1Me by duodenal segments was greater on the day of hatch, decreased until the 2nd wk, and remained constant until adulthood (Figure 1A). In the jejunum, this transport capacity was greater during the 1st wk after hatch, decreased steadily in the 2nd and 3rd wk, and then remained constant until 14 wk (Figure 1B). Total uptake of aGlc1Me by ileal segments was greatest the day after hatch, diminished dramatically during the 1st wk, and remained constant until 14 wk of age (Figure 1C). Phloridzin inhibited more than 85% of the total uptake of monosaccharide in all the segments studied. However, the highest diffusional component was found during the 1st wk posthatch [in duodenum 4.12 ± 0.23; in jejunum 7.42 ± 0.56; in ileum, 4.03 ± 0.35 mmol/100 mg of wet tissue (± SE) after 45 min of incubation]. These values are approximately twice those found in adults.
hatch, the capacities of the jejunum and the ileum were greater than that of the duodenum (P < 0.005) and the intracellular concentration of aGlc1Me in these segments was just over 12 times the concentration of the medium. The duodenum was the intestinal segment with the lowest capacity to transport aGlc1Me at all stages of development (P < 0.005). In 1-wk-old chickens, the jejunum maintained the greatest capacity to accumulate aGlc1Me, but during the 2nd wk this capacity was equal to the ileum, and, after 3 wk, the ileum was the segment with the greatest capacity to accumulate aGlc1Me (P < 0.005). Figure 3 depicts the summed uptake capacity of the whole length of the small intestine as calculated by integrating regional uptakes over the small intestine length and normalized to body weight. Uptake capacity was at a maximum 1 wk after hatch and decreased until adulthood.
Transport of aGlc1Me Figure 2 shows the ability of duodenum, jejunum, and ileum to concentrate aGlc1Me within the cells when the concentration of aGlc1Me in the incubation medium was 0.1 mmol/L and after 45 min of incubation, from the day of hatch to adulthood. Early posthatch, all intestinal regions had higher levels of accumulation than in the other stages of development. After 2 wk in the duodenum and after 3 wk in the jejunum, few changes in this capacity were observed. Shortly after hatch, the capacity of the ileum to accumulate this sugar decreased. Differences in intestinal aGlc1Me transport capacity have been found between the three regions. One day after
FIGURE 2. Intracellular concentration of a-methyl-D-glucoside (aGlc1Me) by phloridzin-sensitive mechanism in everted sleeves from chicken of different ages of duodenum (open bars), jejunum (hatched bars) and ileum (stippled bars) after 45 minutes of incubation. Data are given as means ± SE from 5 to 10 separate experiments per age. Statistical comparisons show that a) in duodenum 1 d > 1 wk > 2 wk > 3 to 14 wk; b) in jejunum 1 d = 1 wk > 2 to 14 wk; c) in ileum 1 d > 1 to 14 wk.
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FIGURE 1. Total uptake of 0.1 mmol/L a-methyl-D-glucoside (aGlc1Me) by chicken everted sleeves from duodenum (A), jejunum (B) and ileum (C) aged 1 d (π), 1 wk (∫), 2 wk (o), 3 wk (◊), 5 to 6 wk (◊) and 12 to 14 wk (…). Phloridzin-insensitive sugar uptake from all the ages are plotted together (ÿ). Data are given as means ± SE from 5 to 10 separate experiments per age. Only standard errors that exceeded size of symbol are shown. Statistical comparison of the curves shows that accumulation: a) in duodenum was 1 d > 1 wk > 2 to 14 wk > phloridzin-treated tissues; b) in jejunum was 1 d = 1 wk > 2 wk > 3 to 14 wk > phloridzin-treated tissues; c) in ileum was 1 d > 1 to 14 wk > phloridzin-treated tissues.
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incubation]. These values are approximately twice those found in adults.
Transport of L-Pro
Total Uptake of L-Pro Figure 4 shows the total uptake of L-Pro 0.1 mmol/L in duodenum, jejunum, and ileum after 45 min of incubation at different ages studied. Total uptake of L-Pro by duodenal segments was greater the day of hatch, decreased until the 3rd wk, and remained constant until adulthood (Figure 4A). In jejunum, this capacity was greater during the 1st wk, decreased until the 5th to 6th wk, and remained constant until 14 wk of age (Figure 4B). Total uptake of L-Pro by ileal segments was maximum the day after hatch, diminished during the 1st wk of age, and remained constant until 14 wk of age (Figure 4C). When Na+ was removed from the incubation medium, the diffusional component was determined. The highest diffusional component was found during the 1st wk [in duodenum 5.40 ± 0.41; in jejunum 5.25 ± 0.29; in ileum, 5.86 ± 0.39 nmol/100 mg of wet tissue (± SE) after 45 min of
FIGURE 4. Total uptake of 0.1 mmol/L L-proline (L-Pro) by chicken everted sleeves from duodenum (A), jejunum (B) and ileum (C) aged 1 d (π), 1 wk (∫), 2 wk (o), 3 wk (◊), 5 to 6 wk (◊) and 12 to 14 wk (»). Na+-independent amino acid uptake from all the ages are plotted together (ÿ). Data are given in means ± SE from 5 to 10 separate experiments per age. Only standard errors that exceed size of symbol are shown. Statistical comparison of the curves shows that accumulation: a) in duodenum was 1 d > 1 wk > 2 wk > 3 to 14 wk = Na+-independent uptake; b) in jejunum was 1 d = 1 wk > 2 wk > 3 wk > 5 to 14 wk > Na+-independent uptake; c) in ileum was 1 d > 1 to 14 wk > Na+-independent uptake.
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FIGURE 3. Summed uptakes capacities of the entire length of the small intestine normalized to body weight for a-methyl-D-glucoside (») and L-proline (…). Data are given in means ± SE. Only standard errors that exceed size of symbol are shown. Statistical comparisons show that for aGlc1Me 1 d < 1 wk > 2 wk > 3 wk > 5 to 6 wk > 12 to 14 wk and for LPro 1 d < 1 wk = 2 wk = 3 wk > 5 to 6 wk > 12 to 14 wk, 1 wk > 3 wk.
Figure 5 shows the ability of the duodenum, jejunum, and ileum to concentrate L-Pro within the cells when the concentration of L-Pro in the incubation medium was 0.1 mmol/L and after 45 min of incubation, from the day of hatch to the adult stage. During the period immediately posthatch, all intestinal regions had higher levels of accumulation than they did during the other stages of development. The duodenum had the lowest cumulative capacity, which was minimal from the 5th to 6th wk of age. No significant changes were observed in the jejunum until the 3rd wk. After the 5th or 6th wk, amino acid was less accumulated. Finally, the ileum had the highest capacity to concentrate L-Pro and presented a different pattern than that of the other two intestinal segments, because the ileum maintained its increased cumulative capacity during the whole period of study. Regional differences in intestinal L-Pro transport capacity were found between the three intestinal regions. One day after hatch, the capacity of the ileum was greater than that of the jejunum and duodenum (P < 0.005) and its intracellular concentration was fivefold greater than the concentration of the medium. This ability was maintained throughout the period studied. During the first 3 wk, the jejunum was able to concentrate double the amino acid present in the incubation medium. From the 5th or 6th wk, this ability diminished significantly. Duodenum was the intestinal segment with the lowest capacity to transport LPro at all stages of development (P < 0.005). Until the 2nd wk, the duodenum accumulated L-Pro but from the 3rd wk only a 5% of the total uptake entered by a Na+ mediated mechanism remained. Figure 3 depicts summed uptake capacity of the whole length of the small intestine. Uptake capacity was lower in adults than in other stages of development. One-week-old
NUTRIENT TRANSPORT IN CHICKEN INTESTINE
chicks showed the greatest uptake capacity for the three segments studied.
DISCUSSION Previous studies on the ontogenesis of nutrient uptake in the intestine of vertebrates suggest that intestinal uptake capacity changes during development and that it is closely matched to intake during postnatal growth (Buddington and Diamond, 1989; Toloza and Diamond, 1990). However, it has been difficult to determine whether the observed changes in the capacity to transport nutrients were due exclusively to the diet or induced by genetic or hormonal factors (Sehata et al., 1984). In the present work, the intestinal transport of nutrients was studied in a precocial species, G. gallus domesticus, raised on a solid diet of constant composition in order to avoid the effects of shifts in diet during the development. However, the results obtained in male White Leghorns may not be considered representative of all breeds and strains of G. gallus domesticus. Previous studies in the small intestine of embryos and newly hatched chicks showed that from at least 2 d before hatching the three regions of the small intestine had a highly developed phloridzin-inhibitable Na+-dependent mechanism to transport aGlc1Me (Esteban et al., 1991). During the embryonic period, the maximal rate of transport significantly increased in all regions with differences between segments (Moreno et al., 1996). Because several controversial points still remain concerning transport during development, we studied aGlc1Me and L-Pro uptake to establish the pattern of changes in the three regions of the small intestine from hatch until the maturity of the chicken. In the present work, the developmental aspects of sugar transport were investigated by an in vitro
technique that represents an integrated system suitable for intestinal segments of small size (Moreno et al., 1996). The variable studied was the time-dependent capacity of the intestinal slices to accumulate substrates. This variable depends, in turn, on the influx and efflux rates as well as on the degree of utilization of the substrate by the intestinal tissue. The selected substrates were aGlc1Me and L-Pro. aGlc1Me is a nonmetabolizable glucose analog that is specific for the apical Na+dependent transporter SGLT1 but not for the other sugar transport systems present in the enterocytes (Kimmich and Randles, 1981; Panayitova-Heiermann et al., 1996). L-Proline is a neutral amino acid largely used in ontogenetic studies (Buddington and Diamond, 1989) and with great importance in chick development (D’Mello, 1994). This study indicated that there was a decrease in the capacity to transport aGlc1Me and L-Pro from hatch to adulthood following separate developmental patterns along the small intestine. Total uptake by duodenal segments was higher on the day of hatch, with a gradual decrease until the 2nd wk for aGlc1Me and 3rd wk for L-Pro, which then remained constant until the adult stage. The jejunum had the highest values during the 1st wk after hatch then decreased until the 3rd wk for aGlc1Me and 5th to 6th wk for L-Pro, remaining constant until the 14th wk. Uptake of the ileum was highest on the 1st d after hatch, diminished shortly after hatch, and remained constant thereafter. Moreover, the duodenum was the intestinal region with the lowest capacity to transport either substrate at all stages of development. Also noteworthy is that from the 5th to 6th wk, the capacity to accumulate L-Pro against a concentration gradient by duodenum virtually disappeared. The jejunum was the segment with the highest capacity to accumulate aGlc1Me during the first 2 wk. From the 3rd wk until the 14th wk, the ileum had the highest capacity to transport aGlc1Me. Furthermore, the segment that had the maximal capacity to accumulate LPro during all the periods studied was the ileum, followed by the jejunum. These variations are consistent with the jejunal sac data of Raheja et al. (1977) and with the brush-border membrane vesicle data of Shehata et al. (1981), but not with the tissue slice data of Bogner and Haines (1964) or Holdsworth and Wilson (1967). Bogner and Haines (1964) and Holdworth and Wilson (1967) authors found no decrease in D-glucose (D-Glc) transport after the 1st wk of life. However, they did not divide the small intestine into regions for their transport studies, whereas we have demonstrated that rates of nutrient transport and developmental timetables vary with the region of the chicken intestine studied (Moreto´ et al., 1991; Amat et al., 1996; Moreno et al., 1996). In a previous study, an age-dependent decline in 3-oxy-methyl-D-glucose (Glc3Me) accumulation by the jejunum and a proximo-distal decrease in cumulative capacity in the cecum has been reported (Planas et al.,
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FIGURE 5. Intracellular concentration of L-proline (L-Pro) by Na+dependent mechanism in everted sleeves from chickens of different ages of duodenum (open bars), jejunum (hatched bars) and ileum (stippled bars) after 45 min of incubation. Data are given as means ± SE from 5 to 10 separate experiments per age. Statistical comparison shows that a) in duodenum 1 d = 1 wk > 2 wk > 3 wk > 5 to 14 wk; b) in jejunum 1 d = 1 to 3 wk > 5 to 14 wk; c) in ileum no differences were found between ages.
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days after hatch, which declined with age as described by other species (Buddington and Diamond, 1989). In conclusion, our results show that the epithelium of the small intestine of the chicken is not homogeneous in its ability to transport nutrients. Furthermore, specific transport mechanisms have displayed different developmental and regional activities. These changes probably represent genomic effects because studies were conducted under conditions in which diet was held constant.
ACKNOWLEDGMENTS We wish to express our sincere appreciation to M. Moreto´ and C. Amat for generous and worthwhile advise as well as for his critical review of the manuscript.
REFERENCES Amat, C., J. M. Planas, and M. Moreto´, 1996. Kinetics of hexose uptake by the small and large intestine of the chicken. Am. J. Physiol. 271:R1085–R1089. Bogner, P. H., and I. A. Haines, 1964. Functional development of active sugar transport in the chick intestine. Am. J. Physiol. 207:37–41. Buddington, R. K., and J. M. Diamond, 1989. Ontogenetic development of intestinal nutrient transporters. Annu. Rev. Physiol. 51:601–619. Buddington, R. K., and J. M. Diamond, 1992. Ontogenetic development of nutrient transporters in cat intestine. Am. J. Physiol. 263:G605–G616. D’Mello, J.P.F., 1994. Amino Acids in Farm Animal Nutrition. CAB International, Wallingford, UK. Esteban, S., M. Moreno, I. Mestre, J. M. Planas, and J. A. Tur, 1991. Regional development of a-methyl-D-glucoside transport in the small intestine of chick embryos and newly-hatched chicks. Arch. Int. Physiol. Biochem. Biophys. 99:425–428. Ferraris, R. P., 1994. Regulation of intestinal nutrient transport. Pages 1821–1844 in: Physiology of the Gastrointestinal Tract. Vol. 2. 3rd ed. L. R. Johnson, ed. Raven Press, New York, NY. Ferrer, R., M. A. Gil, M. Moreto´, M. Oliveras, and J. M. Planas, 1994. Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine. Pflu ¨ gers Arch. 426:83–88. Holdsworth, C. D., and T. H. Wilson, 1967. Development of active sugar and amino acid transport in the yolk sac and intestine of the chicken. Am. J. Physiol. 212:233–240. Kimmich, G. A., and J. Randles, 1981. a-Methyl-glucoside satisfies only Na+-dependent transport system of intestinal epithelium. Am. J. Physiol. 241:C227–C232. Lerner, J., P. H. Burrill, P. A. Sattelmeyer, and C. F. Janicki, 1976. Developmental patterns of intestinal transport mechanisms in the chick. Comp. Biochem. Physiol. 54A: 109–111. Moreno, M., M. Otero, J. A. Tur, J. M. Planas, and S. Esteban, 1996. Kinetic constants of a-methyl-D-glucoside transport in the chick small intestine during perinatal development. Mech. Ageing Dev. 92:11–20. Moreto´, M., C. Amat, A. Puchal, R. K. Buddington, and J. M. Planas, 1991. Transport of L-proline and a-methyl-D-
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1986). The present work shows that there was a progressive decrease in hexose cumulative capacity during development, more significant in the duodenum than in the jejunum and ileum. In 5- to 6-wk-old chickens, the control:phloridzin ratio was around 4 in duodenum, 8 in jejunum, and 17 in ileum. These values maintained the same proportion as the ratios measured in isolated jejunal cells from chickens at the same age, using Glc3Me as substrate (Ferrer et al., 1994). Our results differ from those reported by Obst and Diamond (1992), who found that when D-Glc and L-Pro uptake rates were normalized to wet weight of tissue, there was a peak in uptake for Glc around the 2nd wk and also for L-Pro around the 5th–6th wk after hatch. These peaks have not been observed in tissue accumulation studies, indicating that the increased uptake capacity of the apical mechanism found by the former authors might be counterbalanced by a concomitant increase in the basolateral efflux permeability (Ferrer et al., 1994). In the present study, the diffusional component for aGlc1Me and L-Pro was higher during the 1st wk posthatch. These results agree with those obtained by Shehata et al. (1981) with jejunal slices and Planas et al. (1986) using isolated cells. Va´zquez et al. (1997) showed in chicken jejunal brush-border membrane vesicles that the membrane fluidity was higher from hatch until the 2nd wk of age, followed by a decrease until the 14th wk of age. These changes in membrane lipid composition and fluidity could explain the increase of the diffusional component found in the newly hatched chicks found in this work. Regional studies done in species such as the cat (Buddington and Diamond, 1992) and the bullfrog (Toloza and Diamond, 1990) showed that the uptake capacities of D-Glc and L-Pro decrease from proximal to distal segments. In the chicken, the tissue specific uptake rates of aGlc1Me were higher in the ileum than in the duodenum and the jejunum from the 3rd wk until the 14th wk of age. Also, the uptake rates for L-Pro in the ileum were higher than in the proximal segments from hatch thereafter. These results are consistent with our previous findings in the chicken, in which the ileum had a higher concentrative ability than the duodenum and jejunum for Glc3Me (Ferrer et al., 1994) and with the data of Obst and Diamond (1989) for L-Pro. This can be explained by either a well developed Na+-dependent mechanism in these regions (Obst and Diamond, 1989) or by a low basolateral efflux of the substrate, similar to what was described for glucose analogs in chicken enterocytes (Ferrer et al., 1994). The absolute values of intestinal uptake capacities of the whole length of the small intestine increased with age whereas uptake capacities expressed per gram of body weight showed a large increase during the 1st wk and thereafter a progressive decrease until the 14th wk of age. These results are consistent with higher metabolic rates normalized to body weight during the first
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Raheja, K. L., J. Tepperman, and H. M. Tepperman, 1977. The effect of age on intestinal glucose transport in the chick (Gallus domesticus). Comp. Biochem. Physiol. 58A: 245–248. Shehata, A. T., J. Lerner, and D. S. Miller, 1981. Development of brush-border membrane hexose transport system in chick jejunum. Am. J. Physiol. 240:G102–G108. Shehata, A. T., J. Lerner, and D. S. Miller, 1984. Development of nutrient transport systems in chick jejunum. Am. J. Physiol. 246:G101–G107. Toloza, E. M., and J. M. Diamond, 1990. Ontogenetic development of nutrient transporters in bullfrog intestine. Am. J. Physiol. 258:G760–G769. Va´zquez, C. M., N. Rovira, V. Ruiz-Gutie´rrez, and J. M. Planas, 1997. Developmental changes in glucose transport, lipid composition and fluidity of jejunal BBM. Am. J. Physiol. 273: R1086–R1093. Zivin, J. A., and J. J. Bartko, 1976. Statistics for disinterested scientists. Life Sci. 18:15–26.
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glucoside by chicken proximal cecum during development. Am. J. Physiol. 260:G457–G463. Obst, B. S., and J. M. Diamond, 1989. Interspecific variation in sugar and amino acid transport by the avian cecum. J. Exp. Zool. S3:117–126. Obst, B. S., and J. M. Diamond, 1992. Ontogenesis of intestinal nutrient transport in domestic chickens (Gallus gallus) and its relation to growth. Auk 109:451–464. Panayitova-Heiermann, M., D.D.F. Loo, C. T. Kong, J. E. Levers, and E. M. Wright, 1996. Sugar binding to Na+/ glucose cotransporters is determined by the carboxyterminal half of the protein. J. Biol. Chem. 271: 10029–10034. Planas, J. M., M. Moreto´, E. Gaza, and J. Bolufer, 1982. Changes in intestinal galactose and leucine transport during development in the chick: effect of low external calcium. Poultry Sci. 61:1094–1098. Planas, J. M., M. C. Villa`, R. Ferrer, and M. Moreto´, 1986. Hexose transport by chicken cecum during development. Pflu¨gers Arch. 407:216–220.
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(Key words: White Leghorn, intestinal transport, monosaccharides, development, amino acid) 1998 Poultry Science 77:1347–1353
INTRODUCTION From hatch, the chick intestinal tract undergoes major changes to enable it to extract nutrients from an external solid diet and to absorb them. During this period, there are adaptative changes in gastrointestinal morphology and function that contribute to meeting the increasing metabolic demands during early development (Buddington and Diamond, 1989). Most of the ontogenetic studies on intestinal transport have shown a close relationship between the diet and the capacity to transport nutrients (Ferraris, 1994). However, in most of these studies, the composition of the diet was not kept constant and it is not possible to conclude that the observed changes are due exclusively to feed intake. The process of intestinal development involves such a large number of variables that it is difficult to find the primary cause of age-related changes. In the present study, Gallus gallus domesticus was selected as a useful model for ontogenetic studies
Received for publication October 20, 1997. Accepted for publication May 7, 1998. 1This work was subsidized by grants PB88/0219 and PB91/0159 from Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, Ministerio de Educacio´n y Ciencia, Spain. 2To whom correspondence should be addressed: [email protected]
on intestinal transport because this species can be raised on a diet of constant composition. Studies on the transport of sugars and amino acids in the intestine of chicken during development have been carried out, but the results are contradictory because the separate contributions of multiple mediated pathways were not evaluated, or well-defined regions of the gut were not used (Bogner and Haines, 1964; Holdsworth and Wilson, 1967; Lerner et al., 1976; Raheja et al., 1977; Shehata et al., 1981; Planas et al., 1982 and 1986). In two previous papers (Esteban et al., 1991; Moreno et al., 1996), we have established the developmental patterns and kinetic constants of transport of a-methyl-Dglucoside (aGlc1Me) in the duodenum, jejunum, and ileum of chick during the perinatal period. The important regional and developmental changes found in these studies induced us to extend them until adulthood using not only a monosaccharide but also an amino acid. The purpose of the present study was to determine the capacity to accumulate aGlc1Me and L-Pro as well as to establish the developmental patterns of the mediated and nonmediated component in the different regions of the small intestine of the chicken from the posthatch period until adulthood.
Abbreviation Key: aGlc1Me = a-methyl-D-glucoside; Glc3Me; 3-oxy-methyl-D-glucose; PEG = polyethylene glycol.
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ABSTRACT Development changes in a-methyl-Dglucoside and L-proline accumulation were studied using everted sleeves from the duodenum, jejunum, and ileum of chickens. Six age groups of chickens were used: 1 d and 1, 2, 3, 5 to 6, and 12 to 14 wk. Our results showed the presence of a Na+-dependent mechanism of sugar and amino acid uptake that is already fully developed at hatch. The intestinal transport activity
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Ingredient composition
30.87 26.00 1.50 2.00 3.00 25.80 7.00 0.33 0.05 0.30 2.50 0.50 0.01 0.10 0.04 3,014.00 21.40 0.70 0.35 1.10 0.80 0.25 0.80 0.40
1Mineral premix provides per kilogram of diet: Mn, 50 mg; Zn, 50 mg; Fe, 40 mg; Cu, 5 mg; Se, 0.15 mg. 2Vitamin premix provides per kilogram of diet: vitamin A (as retinyl acetate), 11,250 IU; vitamin E (as dl-a-tocopherol acetate), 32 IU; cholecalciferol, 1,800 IU; thiamine, 5.5 mg; riboflavin, 6.7 mg; pyridoxine, 9.0 mg; cobalamine, 0.01 mg; pantothenic acid, 13.5 mg; niacin, 64.5 mg; folic acid, 1.5 mg; biotin, 0.2 mg.
MATERIAL AND METHODS
Animals Male White Leghorn chickens were used. Animals were obtained from the commercial farm Cooperativa Comarcal d’Avicultura de Reus3 on the day of hatch and maintained under conditions of stable humidity and temperature with a 12:12 h light:dark cycle. The birds had free access to water and feed (Table 1). Experiments were carried out using animals at the following ages: 1 d and 1, 2, 3, 5 to 6, and 12 to 14 wk.
Tissue Uptake of aGlc1Me and L-Pro Birds were killed by decapitation and intestinal segments were removed as follows: duodenal samples were taken from the duodenal loop, jejunal samples were sliced at the level of the yolk stalk, and ileal samples were taken from the end of ileum where the region was connected with mesentery to the ceca. Intestinal segments were trimmed from adherent mesenteric tissue, washed
3Cooperativa Comarcal d’Avicultura, 43280 Reus, Spain. 4NEN Life Science Products, 100 Rue de la Fuse ´ e, B-1130 Brussels,
Belgium. 5Sigma Chemical Co., St. Louis, MO 63178-9916.
Expression of Uptakes Total uptake of aGlc1Me or L-Pro was expressed as nanomoles per 100 mg of tissue and the phloridzinsensitive component of the transport as micromoles per liter of intracellular water.
Materials Labeled compounds [14C(U)]-aGlc1Me, [14C(U)]-L-Pro, and [1,2-3H]-PEG-4000 were purchased from NEN Life Science Products.4 The specific activity and the final concentration of 14C-aGlc1Me were 265 mCi/mmol and 0.04 mCi/mL, respectively. The specific activity and the final concentration of 14C-L-Pro were 290 mCi/mmol and 0.05 mCi/mL, respectively. The specific activity and the final concentration of 3H-PEG-4000 were 8 mCi/mmol and 0.08 mCi/mL, respectively. Unlabeled compounds were obtained from Sigma.5
Statistical Analyses The comparative study of total uptake was performed by fitting the results obtained for each age group and each segment to a horizontal parabola, which was then linearized. The slopes of the resulting lines were compared by ANOVA. Snedecor’s F test was used to detect differences between age groups and intestinal regions (Zivin and Bartko, 1976). A probability level of 5% (P < 0.05) was considered significant.
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Ground yellow corn Wheat Corn gluten feed Corn DDG with solubles Wheat shorts Soybean meal 44 Meat and bone meal Calcium carbonate Dicalcium phosphate Sodium chloride Animal fat Beet molasses L-Lysine·HCl Mineral premix1 Vitamin premix2 Calculated nutrient composition ME, kcal/kg Crude protein, % TSAA, % Met, % Lys, % Thr, % Trp, % Calcium, % Nonphytate phosphorus, %
Percentage
with ice-cold (4 C) saline solution, everted, and cut into segments of about 1 cm. Slices were placed in 10 mL of incubation medium containing (in millimoles per liter): NaCl, 118.5; KCl, 6.2; CaCl 2 , 2.5; KH 2 PO 4 , 1.2; MgSO4·H2O, 1.2; NaHCO3, 24.9, and aGlc1Me or L-Pro, 0.1. The solution was maintained at 37 C, pH 7.4 and bubbled with 95% O2-5% CO2 with adequate stirring. Trace concentrations of 14C-aGlc1Me and 14C-L-Pro were added to quantify the amount of nutrient that was accumulated in the tissue. The passive component for aGlc1Me was determined by adding phloridzin 0.2 mmol/L. For L-Pro, this component was measured by replacing the NaCl with choline chloride in the incubation solution and NaHCO3 with KHCO3. In order to determine the extracellular space, tissue slices were incubated with 3H-polyethylene glycol (PEG)-4000. To calculate the total water content, the intestinal segments were dried at 60 C for 24 h, and the difference between dry weight and total wet weight was determined. Intracellular water of each intestinal region was then estimated by subtracting the extracellular space from the corresponding total water. Tissues were exposed to aGlc1Me or L-Pro at the concentration of 0.1 mmol/L during different times (6, 15, 25, 35, and 45 min). After incubation, the tissues were removed, briefly washed in ice-cold saline, blotted on filter paper, and weighed. Both aGlc1Me and L-Pro were extracted from the tissue using HNO3 0.1 mmol/L overnight at 4 C. Radioactivity in the extract was determined using standard liquid scintillation procedures.
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RESULTS
Total uptake of aGlc1Me Figure 1 shows the total uptake of 0.1 mmol of aGlc1Me/L by pieces of duodenum, jejunum, and ileum at different stages of development during 45 min of incubation. Total uptake of aGlc1Me by duodenal segments was greater on the day of hatch, decreased until the 2nd wk, and remained constant until adulthood (Figure 1A). In the jejunum, this transport capacity was greater during the 1st wk after hatch, decreased steadily in the 2nd and 3rd wk, and then remained constant until 14 wk (Figure 1B). Total uptake of aGlc1Me by ileal segments was greatest the day after hatch, diminished dramatically during the 1st wk, and remained constant until 14 wk of age (Figure 1C). Phloridzin inhibited more than 85% of the total uptake of monosaccharide in all the segments studied. However, the highest diffusional component was found during the 1st wk posthatch [in duodenum 4.12 ± 0.23; in jejunum 7.42 ± 0.56; in ileum, 4.03 ± 0.35 mmol/100 mg of wet tissue (± SE) after 45 min of incubation]. These values are approximately twice those found in adults.
hatch, the capacities of the jejunum and the ileum were greater than that of the duodenum (P < 0.005) and the intracellular concentration of aGlc1Me in these segments was just over 12 times the concentration of the medium. The duodenum was the intestinal segment with the lowest capacity to transport aGlc1Me at all stages of development (P < 0.005). In 1-wk-old chickens, the jejunum maintained the greatest capacity to accumulate aGlc1Me, but during the 2nd wk this capacity was equal to the ileum, and, after 3 wk, the ileum was the segment with the greatest capacity to accumulate aGlc1Me (P < 0.005). Figure 3 depicts the summed uptake capacity of the whole length of the small intestine as calculated by integrating regional uptakes over the small intestine length and normalized to body weight. Uptake capacity was at a maximum 1 wk after hatch and decreased until adulthood.
Transport of aGlc1Me Figure 2 shows the ability of duodenum, jejunum, and ileum to concentrate aGlc1Me within the cells when the concentration of aGlc1Me in the incubation medium was 0.1 mmol/L and after 45 min of incubation, from the day of hatch to adulthood. Early posthatch, all intestinal regions had higher levels of accumulation than in the other stages of development. After 2 wk in the duodenum and after 3 wk in the jejunum, few changes in this capacity were observed. Shortly after hatch, the capacity of the ileum to accumulate this sugar decreased. Differences in intestinal aGlc1Me transport capacity have been found between the three regions. One day after
FIGURE 2. Intracellular concentration of a-methyl-D-glucoside (aGlc1Me) by phloridzin-sensitive mechanism in everted sleeves from chicken of different ages of duodenum (open bars), jejunum (hatched bars) and ileum (stippled bars) after 45 minutes of incubation. Data are given as means ± SE from 5 to 10 separate experiments per age. Statistical comparisons show that a) in duodenum 1 d > 1 wk > 2 wk > 3 to 14 wk; b) in jejunum 1 d = 1 wk > 2 to 14 wk; c) in ileum 1 d > 1 to 14 wk.
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FIGURE 1. Total uptake of 0.1 mmol/L a-methyl-D-glucoside (aGlc1Me) by chicken everted sleeves from duodenum (A), jejunum (B) and ileum (C) aged 1 d (π), 1 wk (∫), 2 wk (o), 3 wk (◊), 5 to 6 wk (◊) and 12 to 14 wk (…). Phloridzin-insensitive sugar uptake from all the ages are plotted together (ÿ). Data are given as means ± SE from 5 to 10 separate experiments per age. Only standard errors that exceeded size of symbol are shown. Statistical comparison of the curves shows that accumulation: a) in duodenum was 1 d > 1 wk > 2 to 14 wk > phloridzin-treated tissues; b) in jejunum was 1 d = 1 wk > 2 wk > 3 to 14 wk > phloridzin-treated tissues; c) in ileum was 1 d > 1 to 14 wk > phloridzin-treated tissues.
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incubation]. These values are approximately twice those found in adults.
Transport of L-Pro
Total Uptake of L-Pro Figure 4 shows the total uptake of L-Pro 0.1 mmol/L in duodenum, jejunum, and ileum after 45 min of incubation at different ages studied. Total uptake of L-Pro by duodenal segments was greater the day of hatch, decreased until the 3rd wk, and remained constant until adulthood (Figure 4A). In jejunum, this capacity was greater during the 1st wk, decreased until the 5th to 6th wk, and remained constant until 14 wk of age (Figure 4B). Total uptake of L-Pro by ileal segments was maximum the day after hatch, diminished during the 1st wk of age, and remained constant until 14 wk of age (Figure 4C). When Na+ was removed from the incubation medium, the diffusional component was determined. The highest diffusional component was found during the 1st wk [in duodenum 5.40 ± 0.41; in jejunum 5.25 ± 0.29; in ileum, 5.86 ± 0.39 nmol/100 mg of wet tissue (± SE) after 45 min of
FIGURE 4. Total uptake of 0.1 mmol/L L-proline (L-Pro) by chicken everted sleeves from duodenum (A), jejunum (B) and ileum (C) aged 1 d (π), 1 wk (∫), 2 wk (o), 3 wk (◊), 5 to 6 wk (◊) and 12 to 14 wk (»). Na+-independent amino acid uptake from all the ages are plotted together (ÿ). Data are given in means ± SE from 5 to 10 separate experiments per age. Only standard errors that exceed size of symbol are shown. Statistical comparison of the curves shows that accumulation: a) in duodenum was 1 d > 1 wk > 2 wk > 3 to 14 wk = Na+-independent uptake; b) in jejunum was 1 d = 1 wk > 2 wk > 3 wk > 5 to 14 wk > Na+-independent uptake; c) in ileum was 1 d > 1 to 14 wk > Na+-independent uptake.
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FIGURE 3. Summed uptakes capacities of the entire length of the small intestine normalized to body weight for a-methyl-D-glucoside (») and L-proline (…). Data are given in means ± SE. Only standard errors that exceed size of symbol are shown. Statistical comparisons show that for aGlc1Me 1 d < 1 wk > 2 wk > 3 wk > 5 to 6 wk > 12 to 14 wk and for LPro 1 d < 1 wk = 2 wk = 3 wk > 5 to 6 wk > 12 to 14 wk, 1 wk > 3 wk.
Figure 5 shows the ability of the duodenum, jejunum, and ileum to concentrate L-Pro within the cells when the concentration of L-Pro in the incubation medium was 0.1 mmol/L and after 45 min of incubation, from the day of hatch to the adult stage. During the period immediately posthatch, all intestinal regions had higher levels of accumulation than they did during the other stages of development. The duodenum had the lowest cumulative capacity, which was minimal from the 5th to 6th wk of age. No significant changes were observed in the jejunum until the 3rd wk. After the 5th or 6th wk, amino acid was less accumulated. Finally, the ileum had the highest capacity to concentrate L-Pro and presented a different pattern than that of the other two intestinal segments, because the ileum maintained its increased cumulative capacity during the whole period of study. Regional differences in intestinal L-Pro transport capacity were found between the three intestinal regions. One day after hatch, the capacity of the ileum was greater than that of the jejunum and duodenum (P < 0.005) and its intracellular concentration was fivefold greater than the concentration of the medium. This ability was maintained throughout the period studied. During the first 3 wk, the jejunum was able to concentrate double the amino acid present in the incubation medium. From the 5th or 6th wk, this ability diminished significantly. Duodenum was the intestinal segment with the lowest capacity to transport LPro at all stages of development (P < 0.005). Until the 2nd wk, the duodenum accumulated L-Pro but from the 3rd wk only a 5% of the total uptake entered by a Na+ mediated mechanism remained. Figure 3 depicts summed uptake capacity of the whole length of the small intestine. Uptake capacity was lower in adults than in other stages of development. One-week-old
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chicks showed the greatest uptake capacity for the three segments studied.
DISCUSSION Previous studies on the ontogenesis of nutrient uptake in the intestine of vertebrates suggest that intestinal uptake capacity changes during development and that it is closely matched to intake during postnatal growth (Buddington and Diamond, 1989; Toloza and Diamond, 1990). However, it has been difficult to determine whether the observed changes in the capacity to transport nutrients were due exclusively to the diet or induced by genetic or hormonal factors (Sehata et al., 1984). In the present work, the intestinal transport of nutrients was studied in a precocial species, G. gallus domesticus, raised on a solid diet of constant composition in order to avoid the effects of shifts in diet during the development. However, the results obtained in male White Leghorns may not be considered representative of all breeds and strains of G. gallus domesticus. Previous studies in the small intestine of embryos and newly hatched chicks showed that from at least 2 d before hatching the three regions of the small intestine had a highly developed phloridzin-inhibitable Na+-dependent mechanism to transport aGlc1Me (Esteban et al., 1991). During the embryonic period, the maximal rate of transport significantly increased in all regions with differences between segments (Moreno et al., 1996). Because several controversial points still remain concerning transport during development, we studied aGlc1Me and L-Pro uptake to establish the pattern of changes in the three regions of the small intestine from hatch until the maturity of the chicken. In the present work, the developmental aspects of sugar transport were investigated by an in vitro
technique that represents an integrated system suitable for intestinal segments of small size (Moreno et al., 1996). The variable studied was the time-dependent capacity of the intestinal slices to accumulate substrates. This variable depends, in turn, on the influx and efflux rates as well as on the degree of utilization of the substrate by the intestinal tissue. The selected substrates were aGlc1Me and L-Pro. aGlc1Me is a nonmetabolizable glucose analog that is specific for the apical Na+dependent transporter SGLT1 but not for the other sugar transport systems present in the enterocytes (Kimmich and Randles, 1981; Panayitova-Heiermann et al., 1996). L-Proline is a neutral amino acid largely used in ontogenetic studies (Buddington and Diamond, 1989) and with great importance in chick development (D’Mello, 1994). This study indicated that there was a decrease in the capacity to transport aGlc1Me and L-Pro from hatch to adulthood following separate developmental patterns along the small intestine. Total uptake by duodenal segments was higher on the day of hatch, with a gradual decrease until the 2nd wk for aGlc1Me and 3rd wk for L-Pro, which then remained constant until the adult stage. The jejunum had the highest values during the 1st wk after hatch then decreased until the 3rd wk for aGlc1Me and 5th to 6th wk for L-Pro, remaining constant until the 14th wk. Uptake of the ileum was highest on the 1st d after hatch, diminished shortly after hatch, and remained constant thereafter. Moreover, the duodenum was the intestinal region with the lowest capacity to transport either substrate at all stages of development. Also noteworthy is that from the 5th to 6th wk, the capacity to accumulate L-Pro against a concentration gradient by duodenum virtually disappeared. The jejunum was the segment with the highest capacity to accumulate aGlc1Me during the first 2 wk. From the 3rd wk until the 14th wk, the ileum had the highest capacity to transport aGlc1Me. Furthermore, the segment that had the maximal capacity to accumulate LPro during all the periods studied was the ileum, followed by the jejunum. These variations are consistent with the jejunal sac data of Raheja et al. (1977) and with the brush-border membrane vesicle data of Shehata et al. (1981), but not with the tissue slice data of Bogner and Haines (1964) or Holdsworth and Wilson (1967). Bogner and Haines (1964) and Holdworth and Wilson (1967) authors found no decrease in D-glucose (D-Glc) transport after the 1st wk of life. However, they did not divide the small intestine into regions for their transport studies, whereas we have demonstrated that rates of nutrient transport and developmental timetables vary with the region of the chicken intestine studied (Moreto´ et al., 1991; Amat et al., 1996; Moreno et al., 1996). In a previous study, an age-dependent decline in 3-oxy-methyl-D-glucose (Glc3Me) accumulation by the jejunum and a proximo-distal decrease in cumulative capacity in the cecum has been reported (Planas et al.,
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FIGURE 5. Intracellular concentration of L-proline (L-Pro) by Na+dependent mechanism in everted sleeves from chickens of different ages of duodenum (open bars), jejunum (hatched bars) and ileum (stippled bars) after 45 min of incubation. Data are given as means ± SE from 5 to 10 separate experiments per age. Statistical comparison shows that a) in duodenum 1 d = 1 wk > 2 wk > 3 wk > 5 to 14 wk; b) in jejunum 1 d = 1 to 3 wk > 5 to 14 wk; c) in ileum no differences were found between ages.
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days after hatch, which declined with age as described by other species (Buddington and Diamond, 1989). In conclusion, our results show that the epithelium of the small intestine of the chicken is not homogeneous in its ability to transport nutrients. Furthermore, specific transport mechanisms have displayed different developmental and regional activities. These changes probably represent genomic effects because studies were conducted under conditions in which diet was held constant.
ACKNOWLEDGMENTS We wish to express our sincere appreciation to M. Moreto´ and C. Amat for generous and worthwhile advise as well as for his critical review of the manuscript.
REFERENCES Amat, C., J. M. Planas, and M. Moreto´, 1996. Kinetics of hexose uptake by the small and large intestine of the chicken. Am. J. Physiol. 271:R1085–R1089. Bogner, P. H., and I. A. Haines, 1964. Functional development of active sugar transport in the chick intestine. Am. J. Physiol. 207:37–41. Buddington, R. K., and J. M. Diamond, 1989. Ontogenetic development of intestinal nutrient transporters. Annu. Rev. Physiol. 51:601–619. Buddington, R. K., and J. M. Diamond, 1992. Ontogenetic development of nutrient transporters in cat intestine. Am. J. Physiol. 263:G605–G616. D’Mello, J.P.F., 1994. Amino Acids in Farm Animal Nutrition. CAB International, Wallingford, UK. Esteban, S., M. Moreno, I. Mestre, J. M. Planas, and J. A. Tur, 1991. Regional development of a-methyl-D-glucoside transport in the small intestine of chick embryos and newly-hatched chicks. Arch. Int. Physiol. Biochem. Biophys. 99:425–428. Ferraris, R. P., 1994. Regulation of intestinal nutrient transport. Pages 1821–1844 in: Physiology of the Gastrointestinal Tract. Vol. 2. 3rd ed. L. R. Johnson, ed. Raven Press, New York, NY. Ferrer, R., M. A. Gil, M. Moreto´, M. Oliveras, and J. M. Planas, 1994. Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine. Pflu ¨ gers Arch. 426:83–88. Holdsworth, C. D., and T. H. Wilson, 1967. Development of active sugar and amino acid transport in the yolk sac and intestine of the chicken. Am. J. Physiol. 212:233–240. Kimmich, G. A., and J. Randles, 1981. a-Methyl-glucoside satisfies only Na+-dependent transport system of intestinal epithelium. Am. J. Physiol. 241:C227–C232. Lerner, J., P. H. Burrill, P. A. Sattelmeyer, and C. F. Janicki, 1976. Developmental patterns of intestinal transport mechanisms in the chick. Comp. Biochem. Physiol. 54A: 109–111. Moreno, M., M. Otero, J. A. Tur, J. M. Planas, and S. Esteban, 1996. Kinetic constants of a-methyl-D-glucoside transport in the chick small intestine during perinatal development. Mech. Ageing Dev. 92:11–20. Moreto´, M., C. Amat, A. Puchal, R. K. Buddington, and J. M. Planas, 1991. Transport of L-proline and a-methyl-D-
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1986). The present work shows that there was a progressive decrease in hexose cumulative capacity during development, more significant in the duodenum than in the jejunum and ileum. In 5- to 6-wk-old chickens, the control:phloridzin ratio was around 4 in duodenum, 8 in jejunum, and 17 in ileum. These values maintained the same proportion as the ratios measured in isolated jejunal cells from chickens at the same age, using Glc3Me as substrate (Ferrer et al., 1994). Our results differ from those reported by Obst and Diamond (1992), who found that when D-Glc and L-Pro uptake rates were normalized to wet weight of tissue, there was a peak in uptake for Glc around the 2nd wk and also for L-Pro around the 5th–6th wk after hatch. These peaks have not been observed in tissue accumulation studies, indicating that the increased uptake capacity of the apical mechanism found by the former authors might be counterbalanced by a concomitant increase in the basolateral efflux permeability (Ferrer et al., 1994). In the present study, the diffusional component for aGlc1Me and L-Pro was higher during the 1st wk posthatch. These results agree with those obtained by Shehata et al. (1981) with jejunal slices and Planas et al. (1986) using isolated cells. Va´zquez et al. (1997) showed in chicken jejunal brush-border membrane vesicles that the membrane fluidity was higher from hatch until the 2nd wk of age, followed by a decrease until the 14th wk of age. These changes in membrane lipid composition and fluidity could explain the increase of the diffusional component found in the newly hatched chicks found in this work. Regional studies done in species such as the cat (Buddington and Diamond, 1992) and the bullfrog (Toloza and Diamond, 1990) showed that the uptake capacities of D-Glc and L-Pro decrease from proximal to distal segments. In the chicken, the tissue specific uptake rates of aGlc1Me were higher in the ileum than in the duodenum and the jejunum from the 3rd wk until the 14th wk of age. Also, the uptake rates for L-Pro in the ileum were higher than in the proximal segments from hatch thereafter. These results are consistent with our previous findings in the chicken, in which the ileum had a higher concentrative ability than the duodenum and jejunum for Glc3Me (Ferrer et al., 1994) and with the data of Obst and Diamond (1989) for L-Pro. This can be explained by either a well developed Na+-dependent mechanism in these regions (Obst and Diamond, 1989) or by a low basolateral efflux of the substrate, similar to what was described for glucose analogs in chicken enterocytes (Ferrer et al., 1994). The absolute values of intestinal uptake capacities of the whole length of the small intestine increased with age whereas uptake capacities expressed per gram of body weight showed a large increase during the 1st wk and thereafter a progressive decrease until the 14th wk of age. These results are consistent with higher metabolic rates normalized to body weight during the first
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Raheja, K. L., J. Tepperman, and H. M. Tepperman, 1977. The effect of age on intestinal glucose transport in the chick (Gallus domesticus). Comp. Biochem. Physiol. 58A: 245–248. Shehata, A. T., J. Lerner, and D. S. Miller, 1981. Development of brush-border membrane hexose transport system in chick jejunum. Am. J. Physiol. 240:G102–G108. Shehata, A. T., J. Lerner, and D. S. Miller, 1984. Development of nutrient transport systems in chick jejunum. Am. J. Physiol. 246:G101–G107. Toloza, E. M., and J. M. Diamond, 1990. Ontogenetic development of nutrient transporters in bullfrog intestine. Am. J. Physiol. 258:G760–G769. Va´zquez, C. M., N. Rovira, V. Ruiz-Gutie´rrez, and J. M. Planas, 1997. Developmental changes in glucose transport, lipid composition and fluidity of jejunal BBM. Am. J. Physiol. 273: R1086–R1093. Zivin, J. A., and J. J. Bartko, 1976. Statistics for disinterested scientists. Life Sci. 18:15–26.
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glucoside by chicken proximal cecum during development. Am. J. Physiol. 260:G457–G463. Obst, B. S., and J. M. Diamond, 1989. Interspecific variation in sugar and amino acid transport by the avian cecum. J. Exp. Zool. S3:117–126. Obst, B. S., and J. M. Diamond, 1992. Ontogenesis of intestinal nutrient transport in domestic chickens (Gallus gallus) and its relation to growth. Auk 109:451–464. Panayitova-Heiermann, M., D.D.F. Loo, C. T. Kong, J. E. Levers, and E. M. Wright, 1996. Sugar binding to Na+/ glucose cotransporters is determined by the carboxyterminal half of the protein. J. Biol. Chem. 271: 10029–10034. Planas, J. M., M. Moreto´, E. Gaza, and J. Bolufer, 1982. Changes in intestinal galactose and leucine transport during development in the chick: effect of low external calcium. Poultry Sci. 61:1094–1098. Planas, J. M., M. C. Villa`, R. Ferrer, and M. Moreto´, 1986. Hexose transport by chicken cecum during development. Pflu¨gers Arch. 407:216–220.
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