We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 (antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein (antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only whenantibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.
- d-glucose transport
- Western blotting
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+-dependentd-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 andd-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 intestinald-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
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 α-methyl-d-glucoside transport. Transport studies were performed in BBMVs using 10 μM α-methyl-d-glucoside (α-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 orAb 2for 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). α-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 nexperiments. Statistical differences were established by Student’st-test with a level of significance ofP < 0.05.
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. Figure1 A 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. Figure1 B shows the relative abundance of protein amounts determined in four assays. However, whenAb 2was used (Fig.2 A), 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. Figure2 B shows the densitometric analysis of four separate assays.
Correlation between maximal transport rates and Western blot analysis. Figure3 shows that there is a linear correlation between the maximal transport rates (V max) for α-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.1 B).
Effect of the presence of antibodies on α-MDG transport. Figure4 shows the effect ofAb 1and Ab 2 on α-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 usingAb 2, no inhibition of transport was observed at any antibody dilution.
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 usingAb 2show 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 theV 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 1shows 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 determinedV 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 thatAb 1recognizes 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 ofAb 1and Ab 2 to interact with α-MDG transport in BBMVs, and the results clearly show that onlyAb 1produces a strong inhibition of apical Na+-dependent hexose influx (Fig.4). Preincubation of the vesicles withAb 2results in low inhibition of α-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 betweenV 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 ofAb 2to recognize the transporter protein.
The enterocytes of the small intestine, the proximal cecum, and the rectum of the chicken have brush-border and basolateral membrane hexose carriers with functional properties similar to those described for mammals (1, 6, 8, 9, 14). Furthermore, the present study also reveals structural similarities in the amino acid sequence between the rabbit SGLT-1 protein and the Na+-d-glucose cotransporter of the chicken, which facilitate identification and quantification of the chicken SGLT-1. However, knowledge of the degree of homology between avian and mammal hexose transporters will only be completed when the cloning and sequencing of the cDNAs encoding chicken SGLT-1 and the GLUTs are achieved.
The rabbit polyclonal antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the rabbit intestinal SGLT-1 sequence and the antigenic peptide were donated by Dr. S. P. Shirazi-Beechey. The rabbit polyclonal antibody raised against the synthetic peptide corresponding to amino acids 564–575 of the rabbit intestinal SGLT-1 sequence and the antigenic peptide were provided by Dr. M. Kasahara and Dr. E. M. Wright, respectively. The authors thank Dr. I. V. Baanante for help and advice.
Address for reprint requests: J. M. Planas, Departament de Fisiologia-Divisió IV, Facultat de Farmàcia, Av. Joan XXIII, s/n, 08028-Barcelona, Spain.
C. Garriga has a grant “Formació d’Investigadors de la Generalitat de Catalunya”. This work was supported by the Ministerio de Educación y Cultura, Spain.
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- Copyright © 1999 the American Physiological Society