Foxl1 null mice have abnormal intestinal epithelia, postnatal growth retardation, and defective intestinal glucose uptake

Jonathan P. Katz,1,2 Nathalie Perreault,1 Bree G. Goldstein,1,2 Hann-Hsiang Chao,2 Ronaldo P. Ferraris,3 and Klaus H. Kaestner1

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 07103–2714

Submitted 29 March 2004 ; accepted in final form 19 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice lacking the mesenchymal winged helix transcription factor Foxl1 exhibit markedly abnormal small intestinal epithelia and postnatal growth retardation. We investigated whether defects in intestinal nutrient uptake and specific transport processes exist in mice homozygous for a Foxl1 null allele (Foxl1–/–). Foxl1–/– mice and controls on a defined genetic background were weighed regularly and killed at 2, 4, and 12 wk of age. Intestinal uptake studies, quantitative real-time PCR, RNase protection assays, and Western blot analyses were performed. Foxl1–/– mice have dysmorphic small intestinal epithelia and postnatal growth retardation. Foxl1–/– mice demonstrate decreased small intestinal uptake of D-glucose in all age groups studied. Intestinal uptake of D-fructose and two amino acids, L-proline and L-leucine, is not altered. Consistent with these findings, Foxl1–/– mice show decreased levels of the intestinal D-glucose transporter SGLT1. Expression of sucrase-isomaltase, lactase, GLUT2, and Na+-K+ ATPase are not changed. Foxl1–/– mice demonstrate markedly abnormal intestinal epithelia, postnatal growth retardation, and decreased intestinal uptake of D-glucose. The specific effect of Foxl1 on intestinal D-glucose uptake is due to decreased production of SGLT1 protein in the small intestine. Thus we identified, for the first time, a link between a mesenchymal factor, Foxl1, and the regulation of a specific epithelial transport process.

knockout mice; intestinal uptake; epithelial-mesenchymal interactions


ONE OF THE MAJOR FUNCTIONS of the small intestine is nutrient absorption. Spatial, temporal, dietary, and endocrine factors all play a significant role in the regulation of nutrient uptake, and thus the control of nutrient absorption may vary greatly, even within a single animal (6). Patterns of gene expression change temporally and along the cranial-caudal and crypt-villus axes of the intestine, contributing to the functional variation of the intestine by age and segment (32). Epithelial-mesenchymal interactions play a critical role in these differentiation processes. Because the epithelial lining is in a state of continuous self-renewal, patterning events along the anterior-posterior and radial axes of the small intestine remain crucial throughout life. Several mesenchymal factors, including Bmp2, Bmp4, Foxl1, Hoxa5, Fgf10, and Nkx2.3, have been shown to affect patterning of gastrointestinal epithelia (4, 16, 24).

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 18–22 (P18–22) 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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. The derivation of the Foxl1–/– mice has been described previously (13). We back-crossed the mutation for eight generations to the C57BL/6 and 129SvEv inbred mouse strains to obtain incipient congenic lines. Mutant and control mice were analyzed as F1 hybrids (C57BL/6 x 129SvEv). The F1 hybrid is a defined genetic background and has the advantage of hybrid vigor by complementation of recessive mutations from parental strains (1). All mice were thus genetically uniform with the exception of the Foxl1 locus. Because previous studies (13) found no difference between Foxl+/+ and Foxl+/– mice, both of these genotypes were used interchangeably as controls. Mice were weighed in the morning for 1 yr after birth. For our experiments, mice were killed at 2, 4, and 12 wk of age to yield time points during suckling, after weaning, and into adulthood.

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 manufacturer’s 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 4–12% 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 {alpha}-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 {alpha}-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 manufacturer’s protocols, counterstained with hematoxylin, dehydrated, and mounted with Cytoseal 60 (Stephens Scientific). Images were captured on a Nikon Microphot FX microscope.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Foxl1/ mice on a defined genetic background exhibit markedly abnormal small intestinal epithelia and severe growth retardation. Foxl1 is a mesenchymal winged-helix transcription factor, the absence of which leads to dramatic alterations in the endodermally derived epithelia of the stomach, small intestine, and colon (13). Initial studies (13), performed on a mixed genetic background, revealed significant variability in the physiological consequences of Foxl1 deficiency. One-third of the Foxl1–/– mice analyzed at the time died within the first week of life after minimal weight gain, and the other two-thirds showed varying degrees of growth retardation. To reduce this variability and to allow for systematic evaluation of the consequences of Foxl1 deficiency, we back-crossed the Foxl1 null allele to two inbred strains of mice, C57BL/6 (B6) and 129 SvEv (129). After eight generations were back-crossed, >99% of all loci are homozygous for the alleles present in the respective parental strains (1).

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.



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Fig. 1. Mice homozygous for a Foxl1 null allele (Foxl1–/–) on an F1 hybrid between the B6 and 129 strains show severe abnormalities in their small intestinal architecture. Hematoxylin and eosin-stained sections of proximal jejunum from 2 (A, B)-, 4 (C, D)-, and 12-wk-old (E, F) wild-type (A, C, E) or Foxl1–/– mice (B, D, F). At 2 wk of age, wild-type mice (A) showed the normal crypt-villus architecture, but Foxl1–/– mice (B) demonstrated distortion of the small intestinal mucosa, with short, broad, and irregular villi. At 4 wk of age, the villi of the Foxl1–/– mice (D) were elongated and the crypt compartment was expanded relative to controls (C). D: mucin-filled cysts (arrowhead) were seen frequently throughout the small intestinal epithelium in the Foxl1–/– mice. By 12 wk of age, the crypt-villus architecture was even more perturbed in the Foxl1–/– mice (F) compared with controls (E). F: mucin-filled cysts (arrowhead) were still seen, along with bifurcated villi (*), crypt fission (arrow), and other abnormalities of villus architecture. Magnification: A, B: x200; CF: x100.

 
Abnormal small intestinal epithelia were seen in the Foxl1–/– mice before weaning, just after weaning, and into adulthood. The number of crypts was also significantly increased in Foxl1–/– mice at 4 and 12 wk of age. By quantitative morphometry, Foxl1–/– mice had 49% more crypts at 4 wk (7.6 ± 0.6 vs. 5.1 ± 0.2 per high-power field) and 22% more crypts at 12 wk (6.0 ± 0.3 vs. 4.9 ± 0.2 per high-power field). As the crypt compartment was markedly expanded in the Foxl1–/– mice, the change in crypt area was likely even greater than that indicated by quantitative morphometry. No changes were seen in the number of crypts at 2 wk of age (4.9 ± 0.2 vs. 4.9 ± 0.3 per high-power field).

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).



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Fig. 2. Foxl1–/– mice are growth retarded. Shortly after birth, Foxl1–/– mice began to lag behind their littermates in weight. Foxl1–/– mice also died at a higher rate postnatally than controls (6 of 14 mutant vs. 6 of 50 wild-type mice by 5 wk of age). Foxl1–/– mice continued to be smaller than controls even after the suckling/weaning transition, but by 12 wk of age, the weights of Foxl1–/– mice and controls were equal. At 1 yr of age, however, Foxl1–/– mice again weighed less than the wild-type mice. These Foxl1–/– mice appeared smaller and visibly sicker than controls. D, day.

 
Foxl1/ mice do not have a delay in the lactase to sucrase transition. The brush-border hydrolases sucrase-isomaltase and lactase are responsible for the cleavage of nonabsorbable disaccharides in the diet to produce monosaccharides that can be absorbed (33). Alterations in dietary intake occur at the suckling-weaning transition, P18–22, coinciding with dramatic changes in gene expression. At this time, the expression of sucrase is markedly induced, whereas lactase expression declines (32).

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.



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Fig. 3. Foxl1–/– mice undergo a normal lactase-to-sucrase transition. A: representative RNase protection assay for sucrase and lactase performed on intestine from 2- and 4-wk-old mice using 20 µg of total RNA revealed a normal lactase to sucrase transition. B: at 2 wk of age, Foxl1–/– mice and controls had low levels of sucrase mRNA. By 4 wk of age, sucrase expression in both groups had markedly increased as expected. C: lactase levels were not significantly different (P = 0.27) between Foxl1–/– mice and controls at 2 wk of age. By 4 wk of age, lactase levels in both groups had declined. For B and C, n = 4 control and 4 mutant animals at each time point.

 
Foxl1/ mice have a specific defect in D-glucose absorption. A thorough understanding of the reasons for growth retardation in the Foxl1–/– mice requires careful analysis of intestinal function, taking into account regional differences in intestinal absorption. The technique of everted intestinal sleeves provides a reproducible method for measuring solute uptake and mucosal permeability in vitro (14). This technique minimizes unstirred layer effects, allows determinations in intestines with small diameters, maintains the tissue in a fixed orientation, and permits extrapolation of summed uptake capacity of the intestine for each nutrient. Whereas methods exist for in vivo study of intestinal absorption, these techniques typically measure uptake of poorly absorbable macromolecules only, thus measuring permeability not absorption, and are less reproducible (2).

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).



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Fig. 4. Foxl1–/– mice demonstrate a significant decrease in the absorption of D-glucose. In 2-wk-old Foxl1–/– mice, D-glucose uptake per unit weight of intestinal tissue was decreased in proximal and midddle small intestine by 10 and 25%, respectively, compared with controls (P = 0.04 by 2-way ANOVA). In mice 4 wk of age, D-glucose uptake was reduced by 38% in the proximal small intestine and 28% in the 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). WT, wild type. *P < 0.05 for region-to-region comparison by 1-way ANOVA.

 
The changes in sugar absorption were specific for D-glucose. Because the absorption of D-glucose was corrected for passive uptake and adherent fluid by using L-glucose, transport measurements reflected only the active transport of D-glucose (14, 15). Moreover, there was no significant change in the uptake of D-fructose, L-proline, or L-leucine in any age group (Table 1), providing additional evidence that the Foxl1 mutation specifically affects active transport of D-glucose. This lack of an efficient uptake mechanism for the most common nutrient, glucose, may contribute to the growth retardation seen during the suckling period in the Foxl1–/– mice (Fig. 2). The mouse undergoes rapid growth during suckling, a time when the diet is high in fat and low in carbohydrate and intake is limited (9), and the ability of the intestine to efficiently digest lactose and absorb glucose is critical (5).


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Table 1. Absorption of D-fructose, L-proline, and L-leucine in the middle small intestine of control and Foxl1–/– mice

 
Foxl1–/– mice demonstrated an adaptive response to the decreased intestinal glucose uptake, increasing their total small intestinal absorptive tissue. By 12 wk of age, wild-type and Foxl1–/– mice weighed approximately the same. However, 12-wk-old Foxl1–/– mice had a significant increase in the amount of tissue per unit intestinal length compared with wild-type mice (39.6 ± 2.2 vs. 33.8 ± 3.1 mg/cm). Such adaptive changes are seen in animals experiencing an increase in metabolic demand (30). Increases in weight per length were seen in all small intestinal segments of the Foxl1–/– mice (proximal: 45.6 ± 5.1 vs. 38.4 ± 3.5 mg/cm; middle 56.3 ± 4.4 vs. 47.2 ± 5.0 mg/cm; distal 34.0 ± 4.1 vs. 28.7 ± 2.7 mg/cm), with P < 0.0001 for these regional effects. Thus the Foxl1–/– mice may be able to overcome the effects of the decreased intestinal glucose uptake through nonspecific increases in mucosal mass. Any remaining deficiencies in intestinal glucose uptake in the older Foxl1–/– mice are less likely to be clinically relevant than in the Foxl1–/– mice experiencing a period of rapid growth (9). These physiological changes correspond with the histological changes seen in Fig. 1.

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 ({alpha}1, {beta}1, {beta}2, {beta}3), demonstrating that of the major constituents of the intestinal glucose transport pathway, only SGLT1 expression was altered.



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Fig. 5. Foxl1–/– mice have decreased protein levels of SGLT1 with normally trafficking to the brush border. A, B: Western blot analyses were performed for SGLT1 using 50 µg of total protein from proximal jejunum of control and Foxl1–/– mice at 4 and 12 wk of age. {alpha}-Tubulin was used as a loading control. A: 4-wk-old Foxl1–/– mice had a trend toward decreased total SGLT1 protein vs. controls (n = 2 control and 2 mutant mice; P = 0.3). B: at 12 wk of age, Foxl1–/– mice showed a significant decrease in expression of SGLT1 protein (n = 4 control and 4 mutant mice; P = 0.02). C, D: immunohistochemistry for SGLT1 in 4- and 12-wk-old mice. C: at 4 wk of age, both control and Foxl1–/– mice have expression of SGLT1 (arrows) at the brush border. D: expression of SGLT1 (arrows) at the brush border is also seen in 12-wk-old control and Foxl1–/– mice (magnification: x200).

 

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Table 2. Expression of GLUT2 and the predominant Na+-K+-ATPase subunits ({alpha}1, {beta}1, {beta}2, {beta}3) in the proximal jejunum of control and Foxl1–/– mice at 12 wk of age TBP served as the internal standard

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The regulation of intestinal glucose transport in vivo is a complex process. Whereas some elements are "preprogrammed," regulation by diet and other factors can occur at certain time points and in certain species. In the mouse, intestinal glucose transport activity and SGLT1 protein expression in immature cells of the small intestinal crypts are programmed irreversibly by dietary carbohydrates (5, 7). Signals to the crypt cells arising from nearby mesenchymal cells could impact the expression of SGLT1 and intestinal glucose transport. Expression of the winged helix transcription factor Foxl1, critical for growth and differentiation of the gastrointestinal epithelium, is localized in the small intestine to the mesenchymal cells adjacent to the crypts (13). Thus Foxl1 is uniquely positioned to impact the glucose transport pathway.

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.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant P30-DK-50306 (to the University of Pennsylvania Center for Molecular Studies in Digestive and Liver Disease) through the Morphology Core and the Molecular Biology Core, NIDDK Grant RO1-DK-53839 (to K. H. Kaestner), an Industry Research Scholar Award from the American Digestive Health Foundation (to K. H. Kaestner), National Science Foundation Grant #IBN-9985808 (to R. P. Ferraris), United States Department of Agrigulture Grant NRI-2000–00876 (to R. P. Ferraris), NIDDK Grant KO8-DK-02809–01 (to J. P. Katz), and a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada BP-220106–1999 (to N. Perreault).


    ACKNOWLEDGMENTS
 
We thank J. Fulmer, K. O’Shea, and S. Dutton Sackett for expert technical assistance.

Current address for N. Perreault: Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, 3001, 12ieme avenue nord, Sherbrooke, Qc, Canada J1H 5N4


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. H. Kaestner, Dept. of Genetics, Univ. of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, PA 19104-6145 (E-mail: kaestner{at}mail.med.upenn.edu)

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.


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