1Department of Genetics and 2Division of Gastroenterology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 3Departments of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 071032714
Submitted 29 March 2004 ; accepted in final form 19 May 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
knockout mice; intestinal uptake; epithelial-mesenchymal interactions
The regulation of intestinal nutrient transport is influenced by a number of factors, including diet, hormones, age, and location, and this regulation is governed by precise patterns of gene expression (5, 10). Developmentally, the suckling-weaning transition between postnatal days 1822 (P1822) in mice represents a critical period during which the intestine undergoes a profound change in gene expression, exemplified by full activation of the gene encoding sucrase-isomaltase (31). Alterations in gene expression along the crypt-villus axis are also crucial to the regulation of nutrient transport. The intestinal glucose transporter SGLT1 and fructose transporter GLUT5, for example, represent contrasting models of regulation along the radial axis. Whereas GLUT5 may undergo de novo mRNA and protein synthesis in differentiated cells of the intestinal villi in response to dietary stimulation, SGLT1 expression is programmed irreversibly in the cells of the intestinal crypts (3, 7). Despite these insights, the molecular mechanisms that control intestinal nutrient uptake are not fully known.
Foxl1 (previously known as Fkh6) (12) is a winged-helix transcription factor required for proper proliferation and differentiation in the gastrointestinal epithelium (11, 13). Foxl1 is expressed in the gastrointestinal tract, specifically in the mesenchyme adjacent to the endodermally derived epithelium. In the small intestine, Foxl1 expression is localized to the mesenchymal cells bordering the crypts. Homozygous null mice for Foxl1/ demonstrate dramatic alterations in the epithelia of the stomach, small intestine, and colon, with severe growth retardation after birth. The changes in the epithelial architecture are the result of a marked increase in the number of proliferating cells in the Foxl1/ mice, a consequence of altered Wnt signaling (22). The etiology of the growth retardation has not been investigated.
Using the everted sleeve method, which determines transport rates across the apical membrane, we explore whether Foxl1/ mice have defects in nutrient transport as a result of the distorted small intestinal epithelial architecture. We show that Foxl1/ mice have a specific decrease in the uptake of the monosaccharide D-glucose in the small intestine, a result of decreased production of the Na+-glucose cotransporter SGLT1 in the small intestine, but no alterations in the uptake of other nutrients. Because nutrient supply in the suckling period is limiting (9), this deficient uptake of D-glucose, the most common nutrient, may contribute to the growth retardation seen in the Foxl1/ mice. Thus we identified, for the first time, a link between a mesenchymal factor, Foxl1, and the regulation of a specific epithelial transport process.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histology. After death, the proximal jejunum was removed and fixed in 4% paraformaldehyde, embedded in paraffin, and 5-µm sections were applied to probe-on plus slides (Fisher). These sections were deparaffinized in xylene, rehydrated in ethanol, and counterstained with hematoxylin and eosin. Sections were then dehydrated in ethanol, cleared with xylene, and mounted with Cytoseal 60 (Stephens Scientific). Images were captured on a Nikon Microphot FX microscope. For quantitative morphometry, crypts per high-power field were counted for 10 high-power fields from four mutant and four control mice.
Intestinal uptake. Mice fed ad libitum were anesthetized by ketamine/xylazine (3.5 ml/kg ip) injection, and a catheter was inserted just distal to the pyloric sphincter. The intestine was cut just proximal to the cecum, and the entire small intestine perfused with cold mammalian Ringer solution. After the mice were killed by transecting the diaphragm, the intestine was rinsed in cold Ringer solution and everted on a metal rod. After the length of the intestine was measured, 1.5-cm pieces were cut from the duodenum and jejunum and slipped over the tip of a smaller metal rod with grooves 1 cm apart. The sleeves of tissue were secured using surgical thread, and excess tissue was removed. The remaining tissue was rinsed in cold Ringer solution and prepared for analysis by preincubating at 37°C for 5 min in Ringer solution bubbled with 95% O2-5% CO2.
Uptake measurements were performed as described (14, 15). For sugars, tissues were incubated (37°C) in oxygenated solutions containing [14C]D-glucose for 1 min or [14C]D-fructose for 2.5 min. [3H]L-glucose was used to correct for adherent fluid and passive diffusion. Hence, only carrier-mediated uptake of D-glucose or D-fructose was determined. The solutions were stirred at 1,200 rpm during the incubation period. The tissues were then rinsed for 20 s in 20 ml of ice-cold Ringer solution to reduce the radioactive label in the adherent fluid. For amino acids, tissues were incubated in [3H]L-proline (2 min) or [3H]L-leucine (2 min), using [14C]polyethylene glycol to correct for adherent fluid. Here, both carrier-mediated and diffusive uptake of L-proline or L-leucine were determined. The uptake rates of nutrients were measured at concentrations yielding Vmax (50 mM). After incubation, the tissues were removed and placed in scintillation vials. Samples were then weighed and solubilized with TS-1 (Research Products International) overnight at 55°C. A liquid-scintillation cocktail, Ecolume (10 ml, ICN), was added, radioactivity was determined by liquid-scintillation spectrometry, and uptake rates were calculated. Statistics were calculated using one-way ANOVA for region-to-region comparisons and two-way ANOVA for uptake along all intestinal regions in each age group.
RNA analyses. RNA was extracted from proximal jejunum using the ToTALLY RNA kit (Ambion) following manufacturers instructions. Ribonuclease (RNase) protection assays were performed using 20 µg total RNA per sample (13). Transcripts were synthesized to yield the following probes: a 123-nt region for lactase using T7 RNA polymerase, a 149-nt region for sucrase using T3 RNA polymerase, and a 501-nt region for GLUT2 using T7 RNA polymerase (17, 20, 26). We employed a 156-nt TATA-box binding protein probe (TBP) as internal standard (26). The RNA fragments obtained were separated on a Novex 6% TBE-urea acrylamide gel (Invitrogen), and the radioactive bands were visualized on a Storm 840 phosphorimager (Molecular Dynamics). Quantitative real-time PCR analysis was performed on a Stratagene Mx4000 Multiplex QPCR System using conditions and primer concentrations suggested by the Brilliant SYBR Green (Stratagene) protocol. Reverse transcription was performed with random hexamers and SuperScript II RT (GIBCO) using RNA from 12-wk-old mice. TBP was used as the internal control.
Protein extraction and Western blot analysis.
Total protein was isolated from proximal jejunum of Foxl1/ mice and controls as described previously (22). Fifty micrograms of total protein were separated on a NuPAGE 412% bis-tris acrylamide gel (Invitrogen) and transferred without methanol to an Immobilon-P membrane for 75 min at room temperature. The membrane was cut and blocked with 5% nonfat dry milk in PBS and 0.1% Tween overnight at room temperature. The membranes were then incubated simultaneously for 3 h at room temperature with SGLT1 rabbit polyclonal IgG (Chemicon) diluted 1:2,000 or -tubulin mouse monoclonal IgG (Sigma) diluted 1:1,000 and then washed 5 times for 10 min in PBS with 0.1% Tween. The membranes were then incubated with anti-rabbit/horseradish peroxidase (HRP) (Amersham Pharmacia Biotech) diluted 1:3,000 (for SGLT1) or anti-mouse/HRP) diluted 1:3,000 (for
-tubulin) for 45 min at room temperature, washed five times for 10 min, and developed with the Enhanced Chemiluminescence Plus Western blot analysis kit (Amersham Pharmacia Biotech).
Immunohistochemistry. After death, the proximal jejunum was snap-frozen in liquid nitrogen, embedded in Optimum Cutting Temperature (Miles Tek), and 5-µm sections were applied to probe-on plus slides (Fisher). Sections were postfixed for 3 min in 10% neutral buffered formalin, washed briefly in tap water, incubated in 1.5% H2O2-95% methanol for 15 min, and washed once in tap water and once in PBS. Nonspecific binding of avidin-biotin was blocked (Avidin/Biotin Blocking Kit, Vector), and slides were washed once with PBS and incubated for 10 min at room temperature with Protein Blocking Agent (Immunotech). SGLT1 rabbit polyclonal IgG (Chemicon), diluted 1:2,500 with 0.1% bovine serum albumin and 0.2% Triton X-100 in PBS (PBT), was applied, and the tissue was incubated overnight at 4°C in a moist chamber. After being washed with PBS, goat anti-rabbit secondary antibody (Vector), diluted 1:200 with PBT, was applied and the tissue was incubated for 30 min at 37°C in a moist chamber. The slides were developed using peroxidase-labeled avidin-biotin complex (Vectastain ABC Kit, Vector) and diaminobenzidine substrate-chromogen solution (Vector) as per the manufacturers protocols, counterstained with hematoxylin, dehydrated, and mounted with Cytoseal 60 (Stephens Scientific). Images were captured on a Nikon Microphot FX microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When either B6 or 129 mice carrying the Foxl1 mutation were bred inter se, most of the resulting Foxl1/ mice died within a few weeks after birth. This increased severity of a targeted mutation in inbred strains of mice has been noted before (21). Next, we analyzed the F1 hybrid between the B6 and 129 Foxl1+/ strains. As expected, Foxl1 mutants on the F1 background were healthier than their counterparts on the parental inbred strains, and the vast majority of Foxl1/ mice on the F1 background survived to adulthood.
Foxl1/ mice displayed severe abnormalities in the architecture of the small intestine, the result of a marked increase in the number of proliferating cells in the Foxl1/ mice (22). At 2 wk of age, wild-type mice (Fig. 1A) showed the normal crypt-villus architecture in the small intestine, whereas Foxl1/ mice had distorted small intestinal mucosa, with short, broad, and irregular villi (Fig. 1B). At 4 wk of age, the villi of the Foxl1/ mice (Fig. 1D) were elongated, and the crypt compartment was expanded relative to controls (Fig. 1C). Mucin-filled cysts (Fig. 1D, arrowhead) were seen throughout the small intestinal epithelia. The crypt-villus architecture was even more perturbed in the Foxl1/ mice at 12 wk of age (Fig. 1F) compared with controls (Fig. 1E). Mucin-filled cysts (Fig. 1F, arrowhead) were still seen, along with bifurcated villi, crypt fission, and other architectural abnormalities. Thus the mesenchymal transcription factor Foxl1 plays an important role in the growth and development of the intestinal epithelium throughout life.
|
On the F1 hybrid background, Foxl1/ mice were born at approximately the normal Mendelian frequency. Shortly after birth, Foxl1/ mice began to lag behind their littermates in weight (Fig. 2). Foxl1/ mice grew more slowly than controls during the suckling period (0.30 ± 0.06 vs. 0.48 ± 0.06 g/day for controls). During the first 2 wk after weaning, Foxl1/ mice gained weight at the same rate as controls (0.66 ± 0.18 vs. 0.60 ± 0.10 g/day for controls), and at 12 wk of age, the weights of controls and Foxl1/ mice were equal (24.2 ± 1.1 vs. 23.5 ± 1.0 g). By 1 yr of age, however, Foxl1/ mice appeared smaller and visibly sicker than controls (40.3 ± 1.6 vs. 33.0 ± 3.4 g), with rectal prolapse and other gastrointestinal tract abnormalities (data not shown). These changes in older mice were likely the result of the impact of Foxl1 on other pathways, including Wnt signaling (22), although nutrient uptake has been shown previously to be more affected in aged mice as well (8).
|
Because an abnormal activation of the disaccharide hydrolases could contribute to the growth retardation in Foxl1/ mice, we investigated whether a delay exists in the suckling-weaning transition. Using total RNA from proximal jejunum of Foxl1/ mice and controls, we performed RNase protection assays for lactase and sucrase (Fig. 3A). Sucrase expression is typically low in preweaning mice. At 2 wk of age, Foxl1/ mice and controls had barely detectable sucrase mRNA levels; by 4 wk of age, sucrase expression was markedly increased (Fig. 3B). Lactase expression was high at 2 wk of age and decreased slightly at 4 wk of age (Fig. 3C). In both preweaning and postweaning mice, there was no significant difference in sucrase or lactase expression between Foxl1/ mice and controls. Thus Foxl1/ mice undergo a normal lactase-to-sucrase transition.
|
We chose to study the sugars D-glucose and D-fructose and the amino acids L-proline and L-leucine (15). D-Glucose is the most common monosaccharide, and its active transport across the brush-border membrane occurs via the intestinal Na+-glucose cotransporter, SGLT1, a carrier shared only by galactose (5). D-Fructose is transported across the brush border by its own "private" carrier, the facilitated fructose transporter GLUT5. L-Proline is a nonessential amino acid that is absorbed predominantly by the imino acid transporter, a carrier specific for proline and hydroxyproline. L-Leucine is an essential, neutral amino acid that shares transporters with other essential and nonessential amino acids (15). Because the majority of nutrient uptake takes place in the duodenum and jejunum, we focused our analyses on these intestinal segments.
Foxl1/ mice demonstrated a significant decrease in the absorption of D-glucose (Fig. 4). In preweaning (2 wk old) Foxl1/ mice, D-glucose uptake per unit weight of intestinal tissue was decreased in proximal and middle small intestine by 10 and 25%, respectively, compared with controls (P = 0.04 by 2-way ANOVA). These decreases in glucose uptake may have been even greater in the preweaning Foxl1/ mice than detected, because these mice had thin, friable small intestines, likely resulting in an artificial elevation in measured glucose uptake in Foxl1/ mice. After weaning, Foxl1/ mice continued to show decreased D-glucose uptake. At 4 wk of age, D-glucose uptake was reduced by 38 and 28%, respectively, in the proximal and middle small intestine of the Foxl1/ mice compared with controls (P = 0.002 by 2-way ANOVA). In adult Foxl1/ mice, 12 wk of age, D-glucose uptake in the proximal and middle small intestine was reduced by 59 and 51%, respectively, vs. controls (P = 0.009 by 2-way ANOVA).
|
|
Foxl1/ mice have decreased protein levels of SGLT1. The Na+-glucose cotransporter SGLT1 is responsible for the vast majority of intestinal D-glucose uptake (35). SGLT1 uses the Na+ electrochemical gradient to drive sugar and water into enterocytes against their concentration gradients (35). D-Glucose completes its journey across the cell into the bloodstream through the facilitative sugar transporter GLUT2 in the basolateral membrane. GLUT2 is also responsible for the transport of D-fructose and D-galactose, and basolateral Na+-K+ pumps generate the electrochemical gradient used for sugar transport (35). Notably, unlike many of the other members of the glucose transport pathway, SGLT1 expression is predominantly regulated at a posttranscriptional level, likely at the level of message stability (19).
To investigate the mechanism of decreased glucose uptake in Foxl1/ mice, we analyzed the expression of the known constituents of the intestinal glucose transport pathway. Foxl1/ mice had lower levels of SGLT1 protein than controls, providing an explanation for the decreased glucose uptake in these mice. At 2 wk of age, SGLT1 was not readily detectable by Western blot analysis (data not shown). By 4 wk of age (Fig. 5A), Foxl1/ mice showed a trend toward decreased SGLT1 protein expression (23% decrease; P = 0.3), and by 12 wk of age (Fig. 5B), Foxl1/ mice had a significant reduction in the levels of SGLT1 protein (63% decrease; P = 0.02). These changes in SGLT1 expression were consistent with the decreases in glucose uptake in the middle small intestine (23 vs. 28% at 4 wk, 63 vs. 51% at 12 wk). By immunohistochemistry, SGLT1 expression was localized to the brush border in both control and Foxl1/ mice at 4 wk (Fig. 5C) and 12 wk of age (Fig. 5D), indicating that whereas Foxl1/ mice had reduced production of SGLT1, transport of the protein to the brush border was not affected. By quantitative real-time PCR (Table 2), Foxl1/ mice had no significant changes in the expression of GLUT2 or any of the predominant Na+-K+-ATPase subunits (1,
1,
2,
3), demonstrating that of the major constituents of the intestinal glucose transport pathway, only SGLT1 expression was altered.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mesenchymal-to-epithelial signaling is critical for patterning along all four major axes of the gut: anterior-posterior, dorsal-ventral, left-right, and radial (16, 24). Endodermally derived signals, from Wnt and hedgehog family members, among others, regulate specific mesodermal targets, impacting multiple aspects of gastrointestinal development, proliferation, and differentiation (23, 29). In the case of Sonic hedgehog (Shh), a vertebrate homolog of Drosophila hedgehog expressed in the endoderm of the gut and its derivatives, its receptor is present only in the gut mesoderm, making epithelial-mesenchymal signaling a requirement for its actions (25, 28). In the mesoderm, Shh induces expression of Bmp4 and other factors leading to patterning events along a number of axes including the radial axis. Mouse knockouts of Shh and Indian hedgehog show abnormalities of the gut, including intestinal transformation of the stomach and reduced epithelial stem cell proliferation and differentiation (23). In this context, Foxl1 has emerged as one of a growing number of mesenchymal factors, including Bmp2, Bmp4, Hoxa5, Fgf10, and Nkx2.3, that play key roles in patterning in the gut (4, 16, 24). Patterning of the epithelium along the radial axis is determined by the regionally associated mesoderm, and, because the gut epithelia undergo continuous self-renewal, such mesenchymal-epithelial interactions remain important throughout life. Interestingly, Foxl1/ mice show reduced expression of both Bmp2 and Bmp4 and altered epithelial Wnt signaling, suggesting non-cell-autonomous mechanisms for the regulation of intestinal epithelial proliferation and differentiation (13, 22).
But why should the control of glucose transport by Foxl1 differ from that of other transport processes? Unlike SGLT1, regulation of expression of the intestinal fructose transporter GLUT5 is rapid (5). Changes in fructose transport are typically paralleled by similar changes in GLUT5 mRNA and protein abundance. Thus any effects of Foxl1 on fructose transport would be rapidly reversed as epithelial cells exit the intestinal crypts, leaving behind the pericryptal mesenchymal cells in which Foxl1 is expressed. Still, the mechanism by which Foxl1 regulates SGLT1 expression remains to be elucidated. Because SGLT1 regulation is predominantly posttranscriptional, it is unlikely that Foxl1 acts at the level of SGLT1 transcription. Foxl1 could, however, alter the expression of PKA and/or PKC, which exert opposing effects on SGLT1 mRNA stability and steady-state levels (27, 36). It has previously been shown (22) that Foxl1 can modulate the levels of heparan sulfate proteoglycans, which are effectors for signaling via Wnt and other secreted ligands. Some Wnt family members act via a noncanonical pathway characterized by the activation of PKC (18), which would serve to decrease the levels of SGLT1 (27). Both decreased SGLT1 and impaired intestinal transport of D-glucose are characteristic of Foxl1/ mice.
Loss of Foxl1 in mice also leads to dramatic perturbation of the epithelia of the small intestine, with postnatal growth retardation. Because nutrient supply in the suckling period is limiting (9), this deficient uptake of D-glucose, the most common nutrient, may contribute to the growth retardation seen in the Foxl1/ mice. Both before and after weaning, Foxl1/ mice demonstrate decreased small intestinal uptake of D-glucose, with no changes in the uptake of D-fructose, L-proline, and L-leucine. Using the inverted sleeve method, we show that this defect occurs at the level of D-glucose transport, downstream of the brush-border hydrolases lactase and sucrase-isomaltase. These changes in D-glucose uptake can be explained entirely by the corresponding decreases in SGLT1.
In addition to a direct effect on D-glucose uptake, other mechanisms may be relevant to the growth retardation in the Foxl1/ mice. Foxl1/ pups exhibit severe structural abnormalities of the stomach and small intestine, with short, broad, irregular villi seen in preweaning mice. These structural abnormalities, both in the gastric and intestinal epithelia, may contribute to alterations in digestion and absorption not identified in these analyses. In addition, lactase levels are decreased by 40% in preweaning mice, although this is not statistically significant in our experiment. Decreased expression or activity of the brush-border hydrolases would further inhibit the ability of Foxl1/ pups to obtain glucose during the critical suckling period. After the suckling-weaning transition, Foxl1/ mice grow at approximately the same rate as controls (0.60 vs. 0.66 g/day for controls) and show signs of adaptation by increasing the intestinal weight per unit length and the total intestinal capacity for D-glucose uptake. Such increases in uptake capacity typically occur in response to increased metabolic demand (30).
It has been demonstrated previously (13, 22) that homozygous null mice for Foxl1 provide an excellent model for characterizing the complex regulatory systems underlying mesenchymal-epithelial signaling. One future area of investigation is the role of dietary regulation of SGLT1 in Foxl1/ mice, because both the activity and expression of SGLT1 normally increase in response to a high-carbohydrate diet (7, 34). Overall, these new data provide compelling evidence that the Foxl1 mutation specifically affects active transport of D-glucose in the small intestine by decreasing production of the Na+-glucose cotransporter SGLT1. Other transport pathways, such as D-fructose, and other components of D-glucose transport, such as GLUT2, are not affected. In conclusion, we identify, for the first time, a link between a mesenchymal factor, Foxl1, and the regulation of a specific epithelial transport process.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
Current address for N. Perreault: Département dAnatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, 3001, 12ieme avenue nord, Sherbrooke, Qc, Canada J1H 5N4
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|