Ontogenetic expression and regulation of Na+-D-glucose cotransporter in jejunum of domestic chicken

Anna Barfull, Carles Garriga, Montserrat Mitjans, and Joana M. Planas

Departament de Fisiologia-Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, E-08028 Barcelona, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To evaluate the effect of age on sugar transport, we determined the uptake of methyl alpha -D-glucopyranoside and the abundance of the Na+-D-glucose cotransporter (SGLT1) in jejunal brush-border membrane (BBM) vesicles of 2-day- and 5-wk-old chickens. Methyl alpha -D-glucopyranoside transport per BBM protein was 40% lower in adults than in newly hatched chickens. This finding was matched by parallel declines in site density of SGLT1, which were detected by Western blot. The immunohistochemical study showed that SGLT1 was exclusively located in the BBM of enterocytes along the entire villus and was absent in the crypt in both age groups, and there was an 11-fold increase in the total absorptive area during development. Northern blot studies of the abundance of SGLT1 mRNA showed similar levels for the groups studied. We conclude that the age-related decline in Na+-dependent hexose transport per unit of BBM protein in the chicken jejunum is due to a reduction in the density of SGLT1 cotransporter and is regulated by a posttranscriptional mechanism.

immunolocalization; Western blot; Northern blot; intestinal hexose transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DURING POSTNATAL DEVELOPMENT in vertebrates, the uptake capacity of the intestine changes according to the nutrient requirements of the organism (6, 20). Specific transport for aldohexoses, normalized to body weight, reaches maximum capacity at birth and then declines throughout development until attaining adult rates of transport in different animal species (2). In most of these studies (20, 22), the composition of the diet was not kept constant, making it difficult to analyze whether the developmental changes were solely due to the diet or caused by other factors. White Leghorn chickens were chosen for this study, since they are dependent on external feed from the day of hatch and can be raised on a diet of constant composition, which makes them a useful model for ontogenetic studies on intestinal nutrient transport.

In chickens, aldohexoses are transported across the brush-border membrane (BBM) of the enterocytes by a Na+-dependent cotransport mechanism similar to that of mammals. This mechanism is sensitive to phlorizin and the membrane electrical potential (8, 10). Na+-D-glucose cotransport is detected in the chicken embryo at the end of incubation, increases until just after hatch, and then declines (18, 25). The peak of sugar absorption around hatching coincides with a complete differentiation of the epithelium (20).

Here, we study the uptake of methyl alpha -D-glucopyranoside, a D-glucose analog specific only for SGLT1, in the jejunum during development and correlate it to the concentration of D-glucose in the luminal content. We used Western blot and immunohistochemical techniques to measure the abundance and distribution of the Na+-dependent hexose transporter. Northern blot assays were performed to determine the specific SGLT1 mRNA abundance.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male White Leghorn chickens (Gallus gallus domesticus L.) were obtained from a commercial farm (Gibert, Tarragona, Spain) on the day of hatch and maintained in standardized temperature and humidity conditions, with a 18:6-h light-dark cycle. The chickens were fed from hatch to 5 wk on a single diet (Gallina Blanca, Purina, Barcelona, Spain) containing (in g/kg diet): 218 crude protein, 33.5 lipid, 375 carbohydrate, and 100.5 crude fiber. The metabolizable energy content was 12.2 MJ/kg diet. Manipulation and experimental procedures were approved by the Ethics Committee of the Universitat de Barcelona.

Experiments were performed on 2-day- and 5-wk-old chickens. Animals were killed by cervical dislocation in the morning, without previous withholding of food.

Morphometrics. Body weight was recorded immediately before each experiment. The jejunum (from the end of the duodenal loop to Meckel's diverticulum) was extracted, freed from adherent mesenteric tissue, and laid flat on wet filter paper to measure the length and perimeter of the relaxed segment. The nominal surface area was measured by placing the jejunum opened lengthwise between two glass plates and outlining the contours on a transparent paper, and the images were then scanned and processed by an image analysis system (IMAT, Serveis Científico-Tècnics, Universitat de Barcelona). Villus length and diameter were measured by light microscopic analysis in jejunal transversal sections from six chickens aged 2 days and six chickens aged 5 wk (details of slide preparation are described in Immunohistochemical localization of SGLT1). Light micrographs showed fingerlike villi, in accordance with Yamauchi and Isshiki (30). In each preparation, the length and diameter of four different villi were measured and the surface area was calculated, with each villus considered to be a cylinder. The effective jejunal surface area was calculated from the nominal surface and the number of villi per surface unit, assuming that the space between villi was negligible. The amplification of the surface produced by microvilli was not considered.

Glucose analysis in luminal content. The jejunal luminal content was drained into a tube kept in crushed ice and immediately centrifuged at 18,000 g at 4°C for 40 min. The supernatant was deproteinized with (in mmol/l) 100 K4[Fe(CN)6] · 3H2O, 250 ZnSO4 · 7H2O, and 100 NaOH to eliminate interfering substances such as hemoglobin, bilirubin, and uric acid and was treated with 1% LiN3 to prevent biological contamination. The sample was again centrifuged at 10,000 g at 4°C for 30 min, and the supernatant was stored at -20°C until analysis. Glucose concentration was measured according to the method of Dahlqvist (5).

Enterocyte isolation. Jejunal enterocytes were isolated as described previously (8) with some modifications. The jejunum was washed in saline buffer and cut lengthwise. Pieces (2 cm each) were placed in a medium containing (in mmol/l) 80 NaCl, 3 K2HPO4, 20 Tris · HCl, 37 mannitol, 0.1 EGTA, and 27 trisodium citrate and 1 mg/ml BSA at pH 7.4. To ensure that cells were obtained from the whole villus including the crypts, the tissue pieces were shaken for 90 min and filtered through a nylon gauze (pore size, 50 µm). The filtrated suspension was centrifuged three times at 800 g at 4°C. The enterocytes obtained were divided into two aliquots to prepare BBM vesicles (BBMV) and extract total RNA in parallel. Cell viability was assessed by trypan blue exclusion.

BBMV preparation and methyl alpha -D-glucopyranoside transport. BBMVs were prepared from isolated jejunal enterocytes by MgCl2 precipitation (10). The pellet was resuspended in a medium containing (in mmol/l) 300 mannitol, 0.1 MgSO4, 0.41 LiN3, and 20 HEPES-Tris (pH 7.4) and adjusted to a final protein concentration of 15-20 mg/ml, as determined by the Coomassie brilliant blue method (1).

The D-glucose analog methyl alpha -D-glucopyranoside was chosen as substrate since it is specific only for SGLT1. Uptake of 0.1 mmol/l methyl alpha -D-glucopyranoside was measured at 37°C by rapid filtration after incubation periods of 5 s and 30 min, in the presence and absence of Na+, to calculate the initial rates and accumulation ratios (10). For each uptake, 10 µl BBMV suspensions were rapidly mixed with 40 µl of the incubation medium containing (in mmol/l) 100 mannitol, 100 NaCl or KCl, 0.1 MgSO4, and 20 HEPES-Tris (pH 7.4) with 0.41 µmol/l LiN3 and an aliquot of methyl alpha -D-[14C]glucopyranoside. At the selected incubation times, the uptake was stopped by the addition of 1 ml of an ice-cold stop solution containing (in mmol/l) 300 mannitol, 0.1 MgSO4, and 20 HEPES-Tris (pH 7.4) with 0.41 µmol/l LiN3. The resulting suspension was rapidly filtered under negative pressure through 0.22-µm pore-size cellulose nitrate filters (Millipore) and washed with 5 ml stop solution. The filters were dissolved in Biogreen-6 cocktail (Scharlau, Barcelona, Spain), and the radioactivity was counted in a Packard TriCarb (model 1500).

Intravesicular volume was calculated under equilibrium conditions as the relationship between the methyl alpha -D-glucopyranoside uptake value at 30 min of incubation and the external concentration of substrate.

Enzyme assays. The purity of the BBMV was determined by assaying for sucrase activity and the ouabain-sensitive Na+-K+-activated ATPase, as described previously (29).

Antibodies and antigenic peptides. Blots and tissue samples were incubated with a rabbit polyclonal antibody, donated by Dr. M. Kasahara (Teikyo University), raised against the synthetic peptide corresponding to amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT1 (13). Purified BBMV from rabbit small intestine was used as a reference material throughout.

In parallel experiments, the antibody was preadsorbed with the corresponding antigenic peptide, provided by Dr. E. M. Wright (University of California, Los Angeles, CA), to confirm specificity of the hybridization.

SDS-PAGE and Western blot analysis. Similar amounts of protein (30 µg) of BBMV were solubilized in Laemmli sample buffer with a final concentration of 23 mmol/l Tris, pH 6.8, 2% SDS, 10% glycerol, 0.001% bromophenol blue, and 5% 2-mercaptoethanol, and resolved by 8% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes in a transfer buffer containing (in mmol/l) 20 Tris and 150 glycine in 20% methanol for 1 h at a constant voltage of 100 V at 4°C, using a transblot apparatus (Bio-Rad, Hercules, CA). Nonspecific binding sites were first blocked with PBS containing 0.05% Tween 20 and 3% BSA. Blots were incubated with the primary antibody at a 1:5,000 dilution for 16 h at 4°C. In simultaneous experiments, nitrocellulose membranes were incubated with the same antibody previously preadsorbed with the antigenic peptide. Membranes were washed (8 times for 4 min each) in PBS containing 0.05% Tween 20. Anti-SGLT1 antibody was detected by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK) using a peroxidase-conjugated anti-rabbit IgG (Sigma Chemical, St. Louis, MO) as a secondary antibody (1:3,000). After detection, hybridization bands were quantified by scanning densitometry.

Immunohistochemical localization of SGLT1. Small fragments of jejunum were fixed in Bouin's solution for 24 h at room temperature, washed, dehydrated in graded ethanol series, and embedded in paraffin wax (Vogel Histo-Comp, melting point 56°C) at 60°C for 20-24 h. Sections (5 µm each) were obtained with a microtome and mounted on slides.

Deparaffinized and rehydrated sections were treated with 3% hydrogen peroxide and 10% methanol in 10 mmol/l PBS (pH 7.2) for 10 min to inhibit tissue peroxidase. Slides were incubated with a rabbit polyclonal anti-SGLT1 antibody (1:150) overnight at 4°C. After being washed in PBS, the sections were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:50) for 1 h and developed with 0.025 mg/ml 3,3'-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide in PBS. Slides were counterstained with Harris hematoxylin solution, dehydrated, cleared in xylene, and mounted in a distyrene, tricresyl phosphate, and xylene synthetic resin.

In parallel experiments, sections were incubated with the antibody previously adsorbed with the corresponding antigenic peptide, to confirm the specificity of the immunostaining obtained.

RNA extraction and Northern blot assays. Whole RNA was extracted from isolated enterocytes using the method of Chomczynski and Sacchi (4). RNA was obtained after phenol extraction and alcohol precipitation and measured by spectrophotometric analysis at 260 and 280 nm to evaluate the purity and concentration of RNA. Samples were loaded in a formaldehyde-agarose gel (15 µg total RNA/lane), stained with ethidium bromide to verify RNA integrity and equivalent gel loading. After electrophoresis (50 V for 5 h), the bands were transferred to a nylon membrane (NytranN, 0.45 µm, Schleicher & Schuell, Dassel, Germany). Membranes were prehybridized (1 h at 68°C) and hybridized (1 h at 68°C) using a commercial solution (ExpressHyb hybridization solution, Clontech, Palo Alto, CA). Blots were washed with 2× SSC-0.05% SDS at room temperature (5 times for 10 min each) and 0.1× SSC-0.1% SDS at 50°C (3 times for 20 min each). Specific RNA was detected with a 3.1-kb EcoRI fragment from pMJC424 plasmid that encodes a rabbit jejunal Na+-D-glucose cotransporter (provided by Dr. E. M. Wright). Probes were labeled with [alpha -32P]dCTP by random priming (random primer DNA labeling mix, Biological Industries, Kibbutz, Israel). Membranes were stripped off and rehybridized with a plasmid encoding for the 18S ribosomal protein. Autoradiographies were carried out at -80°C and quantified by scanning densitometry.

Chemicals. All reagents were obtained from Sigma Chemical except the enhanced chemiluminescence reagents and [alpha -32P]dCTP (sp act, 3,000 mCi/mmol), which were purchased from Amersham International.

Statistics. Results are expressed as means ± SE. Unless otherwise indicated, statistical differences established by Student's t-test are considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight and morphometrics. The body weight of 5-wk-old chickens was 11.5-fold greater than that of the 2-day-old group (Table 1). The jejunum doubled in length and increased 2.5-fold in perimeter during the study period. Jejunal nominal surface area, calculated from intestinal length and perimeter, increased 5.3-fold.

                              
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Table 1.   Body weight and morphometric parameters of jejunum from 2-day- and 5-wk-old chickens

Table 1 also shows the values obtained for villus length and diameter. Villus length increased more than twofold, and the villus diameter increased by 33% from 2 days to 5 wk. Assuming a uniform villus density in each age group, the estimated total villus surface area was eight times higher at 5 wk than at 2 days.

Luminal glucose concentration. The glucose concentration in the lumen increased 48% (P < 0.001) in the jejunum of 5-wk-old chickens (52.5 ± 3.9 mmol/l, n = 8) compared with 2-day-old chickens (27.1 ± 1.9 mmol/l, n = 6).

Characterization of BBMV. In the final BBMV preparation, the sucrase activity was highly enriched in both 2-day- and 5-wk-old groups (13.8 ± 0.9- and 14.4 ± 1.2-fold over the original homogenate, respectively; n = 6), whereas the enrichment of the Na+-K+-ATPase did not increase significantly in any age group (0.8 ± 0.1- and 0.9 ± 0.2-fold, respectively; n = 6). Overall protein recovery, the sum of recoveries in all fractions, was 90.1% ± 3.2 for the 2-day-old group and 94.1% ± 2.9 for the 5-wk-old group.

Na+-dependent uptake of methyl alpha -D-glucopyranoside across BBMV isolated from enterocytes. The initial rate of 0.1 mmol/l methyl alpha -D-glucopyranoside transport across BBMV isolated from jejunal enterocytes of 2-day-old chickens was significantly higher (40%) than in 5-wk-old chickens (35 ± 3 vs. 21 ± 2 pmol · mg protein-1 · s-1, respectively; P < 0.01; n = 6 for each group). The uptake under equilibrium conditions (30 min) was identical in both groups. The resulting accumulation ratios were 5.7 ± 0.8 at 2 days (n = 6) and 3.4 ± 0.6 at 5 wk (n = 6), a reduction of 40% (P < 0.05). No statistical differences were found between the intravesicular volume of 2-day- and 5-wk-old chickens (0.31 ± 0.06 vs. 0.32 ± 0.05 nl/mg protein, respectively; n = 6 for each group).

Immunoblotting. In both groups, the antibody recognized an immunoreactive protein of ~75 kDa. At 5 wk, the relative abundance of SGLT1 was 38% lower compared with the 2-day-old chickens (Fig. 1). When the antibody was preadsorbed with the antigenic peptide, no hybridization signal was detected.


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Fig. 1.   Western blot analysis of SGLT1 in jejunal brush-border membrane vesicles (BBMV) from 2-day- (2d) and 5-wk-old chickens (5w). A: BBMV were incubated with (+) or without (-) the corresponding antigenic peptide. A single 75-kDa band was detected in the absence of the peptide. B: relative abundance of SGLT1 protein measured by optical densitometry. Results are means ± SE of 3 different experiments. * P < 0.01.

Immunohistochemical detection. SGLT1 immunolocalization is shown in Figs. 2 and 3. There was positive staining in villus-attached enterocytes in both 2-day- and 5-wk-old chickens (Fig. 3, A and C). The immunoreaction was uniformly localized in the BBM of the absorptive epithelial cells along the total length of the villus, whereas in mucus-secreting goblet cells no staining was seen. No specific labeling for the SGLT1 protein was seen in crypts (Fig. 3, B and D) or cells from other portions of the tissue, e.g., the lamina propria, the submucosa, or the smooth muscle coat (Fig. 2).


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Fig. 2.   Immunolocalization of SGLT1 protein in jejunal villi from 2-day- and 5-wk-old chickens. Arrowheads indicate that SGLT1 protein is localized in the BBM of the villi of both 2-day- (A) and 5-wk-old chickens (B).



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Fig. 3.   Villus tip and crypt magnification of SGLT1 immunolocalization in jejunum from 2-day- and 5-wk-old chickens. Arrowheads indicate that SGLT1 protein is localized along the entire villus in 2-day- (A) and 5-wk-old chickens (C). In contrast, SGLT1 staining is absent in the crypt in 2-day- (B) and 5-wk-old chickens (D).

No positive staining was observed in the epithelial cells when the tissue slides were preadsorbed with the antigenic peptide (data not shown).

Northern blot. In all cases, the ratio of 260 to 280 nm was >1.8 (data not shown), indicating high purity and low contamination by protein fractions. Hybridization with a specific probe for SGLT1 showed a distinct band of 3.8 kb in both age groups (Fig. 4A). No statistically significant differences were found in SGLT1 mRNA levels between age groups (Fig. 4C). Each well contained equivalent amounts of total RNA, assessed by rehybridizing the same blot with ribosomal 18S RNA (Fig. 4B).


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Fig. 4.   Northern blot analysis of SGLT1 in jejunal enterocytes from 2-day- (2d) and 5-wk-old chickens (5w). A: Northern blot analysis of SGLT1 from isolated enterocytes of 2-day- and 5-wk-old chickens. One transcript of ~3.8 kb was detected in every sample. B: hybridization of the same blot with a probe for 18S ribosomal protein. C: relative abundance of the specific mRNA for SGLT1 normalized by 18S rRNA and measured by optical densitometry. Values are means ± SE of 3 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In chickens, Na+-D-glucose cotransport is already detectable in the intestine during the last days of the embryonic period (18). At hatch, intestinal morphology (16, 30), digestive function (27), and nutrient transport capacity (2) have attained a state of complete development. The maximal capacity per tissue weight to transport monosaccharides is reached the day after hatch and declines afterward until attainment of adult levels at the age of 5 wk (18, 25). This pattern occurs not only in chickens (19, 29) but also in various species of mammals (2, 23).

Consistent with our previous reports (25, 29), in the present study we selected chickens of only two ages: 2 days, representing the newly hatched period when maximal uptake is found, and 5 wk, representing the values attained in adulthood.

The higher ability of the small intestine to transport nonelectrolytes at birth has been explained (24) in terms of the localization of transporters along the whole villus and down into the crypts. Moreover, neither proliferation nor differentiation of enterocytes in chickens is clearly restricted to defined regions of the villus (28). Therefore, to avoid the exclusion of possible functional cells, we isolated enterocytes from both the villus and crypt.

The use of BBMV from the isolated jejunal enterocytes confirmed the results obtained in BBMV isolated from mucosa by Shehata et al. (22) and Vázquez et al. (29). In the present study, methyl alpha -D-glucopyranoside transport per milligram of BBM protein was 40% higher at 2 days than at 5 wk (P < 0.01). This reduction cannot be explained by variations in the purity or size of vesicle preparations, since the transport at equilibrium and the enrichment of enzymatic activities were similar in both groups.

The study of the abundance of the SGLT1 cotransporter using Western blot showed the presence of an immunoreactive band of 75 kDa in both newly hatched and 5-wk-old chickens. The molecular mass was very similar to that in adult chickens (11) and to those reported (13, 23, 26) for SGLT1 in various species. These results indicate that there were no age-related structural changes in SGLT1 that could explain the reduction in methyl alpha -D-glucopyranoside transport.

Densitometric analysis of the immunoblots showed a decline of ~38% in the density of transporters found in the jejunal BBM of adult chickens compared with 2-day-old chickens. This value was well correlated with the decline observed in methyl alpha -D-glucopyranoside transport (40%) and corroborates the decrease in phlorizin binding during chicken development reported by Vázquez et al. (29). Similarly, an age-related reduction in transport rates that could be explained by a decrease in the density of transporters was found in mice (7) and lambs (23).

Shirazi-Beechey et al. (23) suggested that in lambs the decrease observed in transport capacity and SGLT1 expression was regulated directly by luminal D-glucose concentration resulting from dietary changes. In the present study, no dietary changes were introduced, since the diet composition was kept constant from hatch until the 5th wk of age. At 5 wk, chickens had twice the glucose concentration in the luminal content compared with 2-day-old chickens, whereas this finding was reversed for transport capacity. These results suggest that, during the ontogenetic development of intestinal D-glucose transporter, some signal(s) other than substrate concentration is responsible for the control of SGLT1 expression.

Anatomic factors such as nominal surface area and the size of a single villus and microvillus contributed to the D-glucose transport regulation (6). Although there is a lack of agreement on the pattern of development of these parameters in mammals (20), in chickens all reports (16, 30) find these parameters to be increased during development.

Our morphometric study confirmed these results in chickens. Jejunal length and perimeter showed a significant increase that yielded a fivefold enlargement in the nominal surface area in the 5-wk-old chickens compared with the newly hatched chickens. Moreover, the histological study demonstrated a surface enlargement of about eightfold due to villi. This finding was in the range of that described previously by Mitjans et al. (16).

The goal of the next study was to determine the functional surface area involved in the absorption of D-glucose. For this reason, immunolocalization of the Na+-D-glucose cotransporter was carried out. Results indicate that for both groups SGLT1 protein is located on the BBM of mature enterocytes lining the entire villus, whereas it is absent on crypts, goblet cells, and submucosal layers. SGLT1 immunostaining was not seen in the crypts of newborn chickens, indicating that enterocyte differentiation has already taken place when proliferative cells reach the base of the villus, in contrast with the proliferative pattern reported in other studies (24, 28). We conclude that the distribution of SGLT1 in the intestine of both 2-day- and 5-wk-old chickens occurred uniformly along the whole length of the villus, following a similar pattern in both age groups. These results were similar to those found in adult animals by Takata et al. (26) in rat jejunum and Hwang et al. (14) in rabbits but differ from those reported by Haase et al. (12), who found an immunologic reaction in rat crypts using a monoclonal antibody.

The detailed examination of the jejunal epithelium indicates that the ratio between enterocytes and nontransporting cells (e.g., goblet cells) does not change between the two age groups.

Although we have not determined the amplification of the absorptive area caused by microvilli, the results obtained by Mitjans et al. (16) in our laboratory using the same animals enabled us to calculate the total effective area. The microvillus amplification factor was 27.6 and 37.4 for 2-day- and 5-wk-old animals, respectively. On the basis of these values, the total surface area due to villi and microvilli in our study would be 11-fold higher in 5-wk-old chickens than in 2-day-old chickens. This increase in the effective surface area compensates for the decrease in the SGLT1 density observed in 5-wk-old chickens and gives an overall 6.6-fold increase in total SGLT1.

The overall results indicate that the jejunum from newly hatched chickens has a higher transport capacity per BBM protein (40%) than that from 5-wk-old chickens. This cannot be explained by a higher ratio of nontransporting to transporting cells but can be explained by a reduction in the site density of SGLT1 protein.

Finally, the expression of the specific mRNA for SGLT1 was studied to clarify the regulation of the developmental adaptation of D-glucose transport. The 3.8 kb obtained in both 2-day- and 5-wk-old chickens coincided with the 4-kb transcript observed when a homologous chicken probe was used (9).

Previous studies by Lescale-Matys et al. (15) in lambs suggested that the changes in the activity and abundance of the SGLT1 protein observed during development were not directly correlated with the levels of the specific mRNA. Similarly, Miyamoto et al. (17) found that SGLT1 mRNA expression in rats did not vary during development.

The results from this study in chickens demonstrate that SGLT1 mRNA levels are not age dependent. These findings suggest that the changes in transport capacity and density of cotransporters are due to a posttranscriptional regulatory mechanism.

Perspectives. Posttranscriptional regulation of SGLT1 has been described as taking place through different mechanisms, including changes in the mRNA stabilization via protein kinase A activation (21) or changes in the trafficking of the transporter (3). Elucidation of the precise molecular pathways that regulate SGLT1 expression will contribute to the understanding of the mechanisms and signals involved in intestinal development and differentiation.


    ACKNOWLEDGEMENTS

The rabbit polyclonal antibody raised against the synthetic peptide corresponding to amino acids 564-575 of the rabbit intestinal SGLT1 sequence and the antigenic peptide were provided by Drs. M. Kasahara and E. M. Wright, respectively.


    FOOTNOTES

This study was supported by Ministerio de Ciencia y Tecnología Grant AGL2000-0918, Generalitat de Catalunya Grant 1999-SRG-00271 and an FPI grant (C. Garriga), and Ministerio de Educación, Cultura y Deporte FPU grant (A. Barfull).

Address for reprint requests and other correspondence: J. M. Planas, Departament de Fisiologia-Divisió IV, Facultat de Farmàcia, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain (E-mail: jplanas{at}farmacia.far.ub.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00262.2001

Received 15 June 2001; accepted in final form 7 October 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 282(3):G559-G564
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society




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