INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE CHICKEN INTESTINE HAS an apical Na⁺-dependent D-glucose cotransporter (SGLT1) distributed along the small intestine, proximal cecum (10), and rectum (1). It has been identified as a 75-kDa protein designed to transport D-glucose with high affinity (K_m = 0.1 mM) and a turnover number of 2-3 s¹ (12).

It is well known that sugar content of the diet affects both the amount and activity of SGLT1 (7). However, the effect of Na⁺ supply on the intestinal cotransport of Na⁺ and hexoses is still being investigated. It has been shown that a low-Na⁺ diet induces a dramatic reduction in hexose transport in the rectum and the ileum (12) that is rapidly reversed by resalination (13). Because low-Na⁺ intake induces secondary hyperaldosteronism, most of the effects observed are believed to be regulated by aldosterone (14, 20, 26).

In the present study, we examined the effect of changes in dietary Na⁺ intake on the amount of both SGLT1 and SGLT1 mRNA in the chicken small and large intestine to determine whether the reduction in sugar transport is due to changes in the amount of apical SGLT1 and also to know whether this process is controlled at a transcriptional or posttranscriptional level.

	MATERIALS AND METHODS

TOP 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 hatching and were maintained in standardized temperature and humidity conditions, with an 18:6-h light-dark cycle. When the chickens were 10 wk old, they were kept for 14 days on a diet of wheat and barley (1:1). Drinking water contained added NaCl to yield a final concentration of either 0.015 mM [low-salt (LS) diet] or 150 mM [high-salt (HS) diet]. After 2 wk, half of the animals fed a LS diet were switched to a resalination (RS) diet by providing them with drinking water containing 150 mM NaCl. The remaining HS and LS birds were killed after 14 days of diet. Resalinated birds were killed 24 h after resalination.

Serum determinations. Blood was sampled by vein puncture of the wing between 0900 and 1100, and the serum was stored at 20°C. Aldosterone concentration was determined by radioimmunoassay using the Diasorin kit (Vercelli, Italy).

Cell isolation. Enterocytes were isolated from the jejunum, ileum, and rectum of three chickens. The pooled segments were incubated in a medium containing 80 mM NaCl, 3 mM K₂HPO₄, 20 mM Tris · HCl, 37 mM mannitol, 0.1 mM EGTA, 27 mM trisodium citrate, and 1 mg/mL BSA at pH 7.4 (10). Incubation was held for 100 min at 25°C to obtain enterocytes isolated from the entire villus. Cell viability was assessed by Trypan blue exclusion. Enterocytes obtained were divided into two aliquots to prepare brush-border membrane vesicles (BBMVs) and to extract total RNA in parallel form.

BBMV preparation and -methyl-D-glucoside transport. BBMVs were prepared by the MgCl₂ precipitation method (12) from isolated enterocytes. Vesicles were obtained in a medium containing 200 mM mannitol, 50 mM KCl, 0.1 mM MgSO₄, 0.41 µM LiN₃, and 20 mM HEPES/Tris (pH 7.4) and adjusted to a final protein concentration of 15-20 mg/ml. Valinomycin was added to the incubation medium at a final concentration of 45 µM to render the vesicles permeable to K⁺. Uptake of -methyl-D-glucoside (-Glu1Me) was measured by a rapid filtration technique for periods of 5 s and 30 min as described elsewhere (12).

Enzyme and protein determinations. The activity of the ouabain-sensitive Na⁺-K⁺-activated ATPase (Na⁺-K⁺-ATPase, EC 3.6.1.3) and sucrase (EC 3.2.1.48) was routinely assayed as markers of the basolateral (6) and apical membrane (22), respectively. Protein was determined following the method of Bradford (3).

SDS-PAGE and Western analysis. Similar amounts of protein (30 µg) of BBMVs were denatured and resolved by 8% SDS-PAGE. Immunoblotting was carried out as described before (15). Blots were incubated with a rabbit polyclonal antibody raised against the synthetic peptide corresponding to amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT1 (17). In experiments carried out in parallel form, the antibody was preadsorbed with the corresponding antigenic peptide.

RNA extraction and Northern blot analysis. Total RNA was isolated from enterocytes as described previously (5). Each sample was obtained from three separate animals pooled. RNA was quantified by spectrophotometric analysis at 260 nm. Samples were loaded in a formaldehyde-agarose gel (15 µg total RNA/lane) and transferred to a nylon membrane (Nytran 0.45, Schleicher and Schuell, Dassel, Germany). Specific mRNA was detected using a 3.1-kb EcoRI fragment from pMJC424 plasmid encoding a rabbit jejunal Na⁺-D-glucose cotransporter. Probes were labeled with -³²P-dCTP (deoxycytidine triphosphate) by random priming (Random primer DNA labeling mix, Biological Industries, Kibbutz, Israel). Blots were normalized by rehybridization with a plasmid encoding for the 18S ribosomic protein. Autoradiograms were quantified by scanning densitometry.

Chemicals. All unlabeled reagents were obtained from Sigma Chemical (St. Louis, MO) except those used to determine enzymatic activity, which were from Boehringer (Mannheim, Germany). [¹⁴C]--Glu1Me (specific activity 265 mCi/mmol) was purchased from New England Nuclear Research Products (Dreieich, Germany). ³²P-dCTP (specific activity 3,000 mCi/mmol) was purchased from Amersham Ibérica (Madrid, Spain).

Statistical analysis. Results are expressed as means ± SE. Statistical differences were established by ANOVA with a significance level of P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal model. Adaptation to LS diet-induced secondary hyperaldosteronism was maximal after 10 days and maintained until day 14. When animals were switched from the LS to the RS diet, the serum aldosterone concentration decreased to the values characteristic of the HS condition within 24 h (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Evolution of the serum aldosterone concentration in chickens fed high-salt (HS; ), low-salt (LS; ), or resalination (RS; ) diets. Each data point represents means ± SE of 3 animals.

Characterization of the BBMVs. The purity of BBMVs was determined by marker enzyme assays. In the final BBMV preparation, sucrase activity was highly enriched and showed a high overall recovery. The activity of the Na⁺-K⁺-ATPase was not affected. No significant differences were found in enrichments and overall recoveries among the different intestinal segments or diets (Table 1).

Na+-dependent uptake of -Glu1Me across BBMV isolated from enterocytes. Because in previous experiments we studied sugar transport using BBMV from scraped mucosa, we now verified that the BBMV obtained from isolated enterocytes reproduced the pattern (12, 13). The results indicate that the initial rates found were similar to those obtained from mucosa, with values slightly higher due to the enhanced purity of the preparation. In the ileum and rectum, there was a significant reduction in transport in samples from LS diet-adapted chickens compared with values from HS diet animals. No differences were found in jejunal samples between diets. RS restored transport values to the characteristic ones for HS diet. Both the total uptake of 0.1 mM -Glu1Me at equilibrium (30 min) and the mean value of the vesicular volume were identical in all segments studied, and neither was affected by diet (Table 2).

Immunoblots. Figure 2A shows a single band of 75 kDa in jejunal, ileal, and rectal BBMVs that was blocked by preadsorption with the antigenic peptide (Fig. 2B) in agreement with previous observations (15). In HS diet birds, there was no difference in the amount of SGLT1 in the jejunum and the ileum, whereas there was a reduction of 38% in the rectum (Fig. 2C). When animals were switched to a LS diet, the abundance of SGLT1 in the jejunum was unaffected, but it decreased significantly in both the ileum and the rectum, with reductions of 38 and 46% with respect to HS diet values. RS induced a recovery of the amount of SGLT1 in those segments previously affected by the low-Na⁺ intake, to levels similar to those found in HS diet conditions.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   A: Western blot analysis of Na+-dependent D-glucose cotransporter (SGLT1) in brush-border membrane vesicles (BBMVs) obtained from isolated enterocytes of chickens fed HS, LS, and RS diets. In every intestinal segment and for every diet, the antibody recognizes an immunoreactive band of ~75 kDa. B: when the antibody was previously preadsorbed with the corresponding antigenic peptide, no immunoreactive band was detected in any segment. C: graph of the relative abundance measured by optical densitometry. Values represent means ± SE of 3 membrane preparations blotted with BBMV suspensions from different isolations. Statistical differences among segments and diets for SGLT1 relative abundance: HS (H): jejunum (J) = ileum (I) > rectum (R); LS (L): J > I > R; RS (rs): J = I > R; J: ND; I: HS = RS > LS; R: HS = RS > LS. ND, no differences; =, P  0.05; >, P < 0.05.

Correlation between Na+-dependent uptake of -Glu1Me vs. abundance of SGLT1 in BBMV from different regions and diets. Figure 3 shows a linear correlation between the initial rates (in pmol · mg protein¹ · s¹) for -Glu1Me in BBMVs and the relative amount of SGLT1 measured by optical densitometry. It is defined by the equation y = 3.53x + 12.69 (r = 0.9934, P < 0.001).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   -Methyl-D-glucoside (-Glu1Me) uptake vs. relative abundance of SGLT1 protein in BBMVs from J, I, and R of chickens fed a HS (H), LS (L), or RS (rs) diet. Values represent means ± SE of 3 BBMV or membrane preparations. Correlation between uptake and optical densitometry is defined by the equation y = 3.53x + 12.69 (r = 0.9934, P < 0.001).

Specific mRNA abundance. The ratios of absorbance at 260 and 280 nm in the solutions of total RNA were higher than 1.8 (data not shown), indicating a high purity and low contamination by protein fractions. Hybridization with a specific SGLT1 probe showed a single band of 3.8 kb in every intestinal segment (Fig. 4A). Rehybridization of the same blot with a ribosomal probe (Fig. 4B) was aimed at normalizing mRNA levels with respect to a similar amount of total RNA loaded per well. There were no regional or dietary differences in the amount of SGLT1 mRNA.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   A: Northern blot analysis of SGLT1 from isolated enterocytes of chickens fed HS, LS, and RS diets. A single 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 represent means ± SE of 3 membrane preparations using total RNA extracted from different enterocyte suspensions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A loading or depletion of dietary Na⁺ affects the intestinal cotransport of Na⁺ and hexoses in the chicken distal intestine (2, 12, 13, 18). To study the effects of Na⁺ intake on the expression of SGLT1 protein and mRNA, we isolated enterocytes from the jejunum, ileum, and rectum of chickens fed a HS, LS, and RS diet that were used 1) to prepare BBMVs for Western blot and transport studies and 2) to extract total RNA. The use of enterocytes instead of mucosa was chosen to avoid interferences in the extraction and detection of mRNA caused by nonepithelial cells.

The results obtained in transport studies confirm that adaptation to the LS diet reduced -Glu1Me transport in ileum (42%) and rectum (51%) BBMVs and that RS reversed it rapidly, confirming previous results (13).

The reduction in the absorption rate of a nutrient is an adaptive process that often occurs through changes in the number of transporters per cell. Abundance of SGLT1 in BBMV was investigated by Western blot analysis. Hybridization with the specific antibody showed the presence of a single band of 75 kDa in all three intestinal segments. There were regional differences in the amount of SGLT1, with a pronounced decrease observed in the rectum, as has been previously described (2, 15). LS diet had no effect on the abundance of SGLT1 in the jejunum. However, in the more distal intestinal segments, LS diet adaptation induced a reduction of 38% in the ileum and of 46% in the rectum. These reductions were similar to those observed in the adaptation of -Glu1Me transport to Na⁺ depletion. When -Glu1Me transport and the amount of SGLT1 were analyzed together, a highly significant linear correlation was found, indicating that the changes in transport induced by the adaptation to Na⁺ intake were due to variations in the number of transporters. These results were consistent with those obtained by measuring the phlorizin-specific binding as an estimate of the number of hexose transporters (13) and with the results obtained in the hen rectum by Bindslev et al. (2) using the Western blot technique. The failure of other authors (8) to find SGLT1 in the rectum can be attributed to the antibody used (15).

Aldosterone seems to be involved in the regulation of intestinal transport in response to dietary Na⁺: in LS diet birds, plasma aldosterone was five- to eightfold higher than in the HS diet (12, 14) and recovered in only 4 h after RS (13). The effects on hexose transport and SGLT1 abundance are closely related to changes in the serum aldosterone concentration (13), and chickens fed a HS diet have LS diet transport values when aldosterone is administered subcutaneously. These results point to aldosterone as being the signal that controls hexose transport in Na⁺ depletion (14).

As the effects of aldosterone are mediated by a wide range of cellular pathways (28), the mechanism by which it regulates hexose transport is unknown. In the present study, Northern blots were performed to assess whether transcription of the SGLT1 gene is affected by the hyperaldosteronism established after adaptation to Na⁺ depletion. It is known that regulation of SGLT1 in response to developmental or dietary changes occurs without significant variations in the levels of specific mRNA (21, 27). These studies suggest that adaptive changes of this transporter are regulated through variations in the dietary content (7, 21, 24). However, in our study, both groups of animals were fed diets containing equivalent amounts of carbohydrates, and there were no differences in the consumption of food nor in body weight, as was previously reported (12). This indicates that the signal that controls hexose transport capacity in the distal intestine is not dependent on the luminal glucose concentration.

The luminal concentration of Na⁺ itself cannot account for all the adaptive changes observed in LS diet-fed animals. Although there is a lower concentration of this ion all through the gut of LS diet birds (data not shown), only in the ileum and rectum were there evident decreases of SGLT1 expression, whereas the jejunum remained unaffected. These results suggest that the signal that controls SGLT1 expression might be not luminal but secondary to diet-induced changes in circulating aldosterone (20). Supporting this view, a recent study demonstrated that aldosterone itself was able to mimic the effects of a low-Na⁺ diet (14, 20) with no changes in the luminal Na⁺ content.

SGLT1 mRNA was detected in all three intestinal segments of the chicken without regional differences, and it showed a single transcript of 3.8 kb, as found by Gal-Garber et al. (11). This is the first time that SGLT1 mRNA is reported in the chicken rectum. The fact that other authors did not find significant levels of either mRNA or SGLT1 expression in the chicken rectum (9) should be attributed to methodological reasons, such as the use of whole mucosa instead of isolated enterocytes.

Having established that mRNA for SGLT1 could be found in each intestinal segment, we examined the effect of changes in the dietary sodium content on SGLT1 mRNA.

Results clearly show that the amount of specific mRNA does not vary significantly between diets in any intestinal segment. Therefore, the observed changes in transport and in the number of transporters cannot be explained by induction or repression of the SGLT1 gene, suggesting that SGLT1 is not regulated transcriptionally. The role of aldosterone in the regulatory network might be ascribed to a posttranscriptional control. In support of this hypothesis, a mineralocorticoid response element has not yet been found in the SGLT1 promoter, whereas it has been found in other promoters of membrane transporters such as the human Na⁺-K⁺-ATPase (19).

Perspectives

An LS diet induces in the chicken rectum a higher expression of those proteins involved in Na⁺ absorption in the intestine (8, 16, 26). Although it has been speculated that switching from SGLT1 synthesis (in the HS condition) to the ENaC, Na⁺/H⁺ exchanger isoform 2, and Na-K-ATPase (in the LS condition) is due to an energy-saving response, the physiological advantage of a SGLT1 downregulation remains unclear. Further experiments regarding the precise molecular pathways that regulate both Na⁺ and glucose transporters in the intestine, especially those triggered by aldosterone, would contribute to the understanding of the mechanisms responsible for the control of the homeostasis of sodium.