INTRODUCTION

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THE CHICKEN INTESTINE transports aldohexoses by mechanisms similar to those described for mammals. The enterocytes from the small and large intestine have an apical Na⁺-dependent D-glucose cotransporter that is sensitive to phlorizin and to the membrane electrical potential difference (1, 6, 7, 14) and a basolateral, GLUT-2 carrier, transporting D-glucose and D-fructose with low affinity and high capacity (8, 15).

The distribution of hexose carrier systems in the intestinal epithelium is not limited to the small intestine but extends to regions of the large intestine such as the proximal cecum and the rectum (1, 6). In all regions studied, the apical carrier has substrate specificity (14), kinetic constants, and phlorizin binding properties (7) that point to identifying this system with the mammalian SGLT-1 (10). With the use of antibodies raised against the rabbit SGLT-1, a 64-kDa protein was identified in the small intestine (4, 5). However, the study of the molecular biology of the chicken rectal glucose transporter has yielded conflicting results. On the one hand, Bindslev et al. (2) identified a 70- to 80-kDa band in scrapings of rectal mucosa corresponding to SGLT-1. On the other, Donowitz et al. (4), although describing D-glucose transport in the rectum, were not able to correlate this activity with SGLT-1 protein.

In the present report we identify the chicken intestinal D-glucose transport with the presence of SGLT-1 both in the small and the large intestine and conclude that the conflicting results from other laboratories can be explained by differences in the specificity of antibodies employed.

	MATERIAL AND METHODS

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Animals. Male White Leghorn chickens (Gallus gallus domesticus L.) were obtained from a commercial farm (Gibert, Tarragona, Spain) the day of hatch and maintained in standardized temperature and humidity conditions with a 18:6-h light-dark cycle. The birds had free access to water and a diet containing (in g/kg diet) 107.9 crude protein, 20.5 lipid, 626.4 carbohydrate, and 100.5 crude fiber. Manipulation and experimental procedures are in accordance with the Spanish regulations for the use and handling of animals for experiment.

Brush-border membrane vesicles preparation. Experiments were carried out with 12-wk-old chickens. Brush-border membrane vesicles (BBMVs) were prepared by an MgCl₂ precipitation method (18).

Protein and enzyme assays. Protein was measured using the Coomassie brilliant blue method (3). To guarantee the purity of the BBMVs, the enrichment in the activity of sucrase and the ouabain-sensitive Na⁺-K⁺-activated ATPase were routinely checked as described before (18).

Antibodies and antigenic peptides. Blots were incubated with two different antibodies against SGLT-1. Antibody 1 (Ab 1) was a rabbit polyclonal antibody (donated by Dr. M. Kasahara) raised against the synthetic peptide corresponding to amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 (10). Antibody 2 (Ab 2) was a rabbit polyclonal antibody (provided by Dr. S. P. Shirazi-Beechey) raised against the synthetic peptide corresponding to amino acids 402-420 of the rabbit intestinal SGLT-1 sequence (10). Purified BBMVs from rabbit small intestine possessing the SGLT-1 cotransporter protein (12) were used as a reference material throughout.

In experiments carried out in parallel, the antibodies were preabsorbed with the corresponding antigenic peptide corresponding to amino acids 564-575 and 402-420 provided by Dr. E. M. Wright and Dr. S. P. Shirazi-Beechey, respectively.

SDS-PAGE and Western analysis. Similar amounts of protein (30 µg) of BBMVs were solubilized in Laemmli sample buffer and resolved by 8% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes for 1 h at constant voltage of 100 V. Immunoblotting and visualization of particular proteins by immunoreactivity was carried out as described previously by Bindslev et al. (2) when Ab 1 was used or as described by Pajor et al. (17) when Ab 2 was used.

Effect of the presence of antibodies on -methyl-D-glucoside transport. Transport studies were performed in BBMVs using 10 µM -methyl-D-glucoside (-MDG) as substrate (10 s incubation, 37°C) and a rapid filtration technique, as previously described (9). The vesicles were preincubated with either Ab 1 or Ab 2 for 30 min at 37°C. Each antibody was added at three final dilutions, 1:500; 1:1,000, and 1:2,000. The use of different dilutions was decided because the titre of antibodies in the rabbit serum was not known. We also decided to use an antibody concentration higher than that used in the Western blot analyses to guarantee that enough antibody was present to bind the SGLT-1 protein.

Chemicals. All unlabeled reagents were obtained from Sigma Chemical (St. Louis, MO), except the enhanced chemiluminiscence reagents, which were from Amersham International (Buckinghamshire, UK). -Methyl-D-[¹⁴C]glucoside (specific activity 265 mCi/mmol) was purchased from New England Nuclear Research Products (Dreieich, Germany). The final activity of labeled substrate in the incubation medium was 2.65 µCi/ml.

Statistical analysis. Results were expressed as means ± SE of n experiments. Statistical differences were established by Student's t-test with a level of significance of P < 0.05.

	RESULTS

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Characterization of the BBMVs. The purity of BBMVs was determined by marker enzyme assays. In the final BBMV preparation, the activity of sucrase was highly enriched (14.1 ± 1.1-fold over the original homogenate; n = 6) and the activity of the Na⁺-K⁺-ATPase, a marker of the basolateral membrane, was not enriched (0.8 ± 0.1-fold; n = 6).

Immunoblots. Figure 1A shows an experiment carried out using Ab 1. This antibody recognizes a single band of 75 kDa in jejunal, ileal, and rectal BBMVs that was blocked by preabsorbing the antibody with the antigenic peptide. Figure 1B shows the relative abundance of protein amounts determined in four assays. However, when Ab 2 was used (Fig. 2A), two bands, one of 64 and another of 35 kDa, were detected in the vesicles from the jejunum and the ileum; however, in the vesicles from the rectum no signal has been found. Figure 2B shows the densitometric analysis of four separate assays.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   A: Western blot analysis of SGLT-1 in brush-border membrane vesicles (BBMVs) of rabbit small intestine (lanes 1 and 5) and in BBMVs from the jejunum (lanes 2 and 6), ileum (lanes 3 and 7), and rectum (lanes 4 and 8) of chicken. Samples (30 µg of protein/lane) were blotted using a rabbit polyclonal antibody raised against synthetic peptides corresponding to amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT-1, in presence (lanes 1-4) and in absence (lanes 5-8) of the antigenic peptide. B: relative abundance measured by optical densitometry. Results show means ± SE of 4 different experiments. Molecular mass standard is shown on left. * Significantly different (Student's t-test, P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   A: Western blot analysis of SGLT-1 in BBMVs from the jejunum (lanes 1 and 4), ileum (lanes 2 and 5), and rectum (lanes 3 and 6). Samples (30 µg of protein/lane) were blotted using a rabbit polyclonal antibody raised against synthetic peptides corresponding to amino acids 402-420 of the deduced amino acid sequence of rabbit intestinal SGLT-1, in presence (lanes 1-3) and in absence (lanes 4-6) of the antigenic peptide. B: relative abundance measured by optical densitometry of 64-kDa band (open bars) and 35-kDa band (hatched bars). Results show means ± SE of 4 different experiments. Molecular mass standards are shown on left. * Significantly different (Student's t-test, P < 0.05).

Correlation between maximal transport rates and Western blot analysis. Figure 3 shows that there is a linear correlation between the maximal transport rates (V_max) for -MDG, previously determined by Garriga et al. (9) in BBMVs and the relative abundance of SGLT-1 determined by densitometric analysis of Western blots carried out using Ab 1 (from Fig. 1B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   -Methyl-D-glucoside (-MDG) uptake vs. relative abundance of SGLT-1 protein in BBMVs from jejunum (J), ileum (I), and rectum (R). Maximal transport rates (Vmax) are from Garriga et al. (9); relative abundance of SGLT-1 was measured by optical densitometry. Results are means ± SE of 3 or 4 separate experiments. Correlation between Vmax and optical densitometry is defined by the equation y = 3.75x  91.32 (r = 0.9903).

Effect of the presence of antibodies on -MDG transport. Figure 4 shows the effect of Ab 1 and Ab 2 on -MDG transport in BBMVs. In vesicles preincubated with Ab 1, a strong inhibition (>88%) of the apical Na⁺-dependent hexose transport was found in all three segments. However, in the vesicles preincubated with Ab 2, much lower inhibitory effects were observed; in the jejunum and ileum, the maximal inhibition of transport obtained was 25% (1:1,000, 1:500 dilutions), whereas in the rectum, where no signal was found in Western blots using Ab 2, no inhibition of transport was observed at any antibody dilution.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effect of preincubation of BBMVs with antibodies 1 and 2 raised against rabbit SGLT-1 (see MATERIAL AND METHODS for further details) on Na+-dependent -MDG transport. Vesicles from the jejunum, ileum, and rectum were preincubated for 30 min at 37°C. Each antibody was added at final dilutions of 1:500 (A), 1:1,000 (B), and 1:2,000 (C). Results (mean ± SE of 3 different experiments) are expressed as percentage of -MDG uptake at 10 s in BBMVs preincubated without antibodies. Ct and Ctl, control.

	DISCUSSION

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In a previous study, Vázquez et al. (19) reported that a polyclonal antibody to the synthetic peptide corresponding to amino acids 402-420 of the rabbit SGLT-1 sequence recognizes an immunoreactive protein of 64 kDa in BBMVs of chicken jejunum. Subsequently, Dyer et al. (5) and Donowitz et al. (4), using an analogous antibody, identified a similar immunoreactive protein in other regions of the small intestine. The antibody used (Ab 2) was synthesized against a hydrophilic, accessible region of the membrane protein in accordance with the different models proposed for the rabbit SGLT-1 (10, 11).

However, the results obtained in our laboratory using Ab 2 show that this antibody reacts with the brush-border membrane of the jejunum and ileum, recognizing two bands rather than one. Furthermore, both bands can be blocked by preabsorbing the antibody with the antigenic peptide. We have also observed that the BBMVs from the chicken rectum do not show any immunoreactive bands. These results indicate that the Ab 2 antibody can react with at least two kinds of proteins from the brush-border membrane of the chicken small intestine. Moreover, when the values of blot densitometry obtained using this antibody and the V_max determined in different intestinal regions (9) are compared, no correlation between density of transporter and capacity to transport aldohexoses is found.

Another polyclonal antibody, prepared against the synthetic peptide corresponding to amino acids 564-575 of the rabbit SGLT-1 sequence (Ab 1), was then assayed. This antibody recognizes a hydrophilic, accessible region of the rabbit SGLT-1 protein that is located in an intracellular loop (10, 11). Figure 1 shows that, with this antibody, a single 75-kDa immunoreactive protein can be recognized that is blockable by preabsorbing the antibody with the antigenic peptide. Results of blot densitometry are well correlated with the previously determined V_max of phlorizin-sensitive Na⁺-D-glucose uptake (Ref. 9; Fig. 3), supporting the view that this protein is an SGLT-1 cotransporter or a closely related protein. In addition, we have demonstrated by immunohistochemical localization that Ab 1 recognizes a protein located exclusively in the brush border of villus enterocytes without reacting with other cell types (16).

Bindslev et al. (2), using antibodies raised against the rabbit SGLT-1 peptide sequence 564-575, were also able to identify in the hen rectum a 75-kDa protein that correlated well with functional expression of phlorizin-sensitive Na⁺-D-glucose cotransport. This was not confirmed by Dyer et al. (5), because they reported expression of SGLT-1 in chicken duodenum, jejunum, and ileum but no specific immunoreactive bands were detected in membranes prepared from the rectum; moreover, because no SGLT-1-related cDNA could be identified in the rectum (4, 5) they concluded that the Na⁺-dependent glucose transport in this region should not be attributed to an SGLT-1 type protein.

In the present study we tested the capacity of Ab 1 and Ab 2 to interact with -MDG transport in BBMVs, and the results clearly show that only Ab 1 produces a strong inhibition of apical Na⁺-dependent hexose influx (Fig. 4). Preincubation of the vesicles with Ab 2 results in low inhibition of -MDG uptake in the small intestine and has no effect in the rectum, the segment where no signal was found in Western blots using Ab 2. The lack of correlation between V_max and the optical density of blots using Ab 2, together with the failure to recognize SGLT-1 protein in the chicken rectum, suggests either that there are species and regional differences in the 402-420 amino acid sequence of the SGLT-1 or that Ab 2 reacts with a brush-border protein not related to SGLT-1 activity.

We conclude that the antibody against the amino acid sequence 564-575 of the rabbit SGLT-1 recognizes the glucose transporter present in both small and large intestine of the chicken. Moreover, the relative protein amounts found by Western blot correlate well with the functional expression of phlorizin-sensitive Na⁺-D-glucose cotransporter in all intestinal regions. The conflicting results from other laboratories in the identification of SGLT-1 in distal intestine of the chicken may be ascribed to the failure of Ab 2 to recognize the transporter protein.

Perspectives