The roles of glucagon-like peptide-2 and the intestinal epithelial insulin-like growth factor-1 receptor in regulating microvillus length

Microvilli are tiny projections on the apical end of enterocytes, aiding in the digestion and absorption of nutrients. One of their key features is uniform length, but how this is regulated is poorly understood. Glucagon-like peptide-2 (GLP-2) has been shown to increase microvillus length but, the requirement of its downstream mediator, the intestinal epithelial insulin-like growth factor-1 receptor (IE-IGF-1R), and the microvillus proteins acted upon by GLP-2, remain unknown. Using IE-IGF-1R knockout (KO) mice, treated with either long-acting human (h) (GLY2)GLP-2 or vehicle for 11d, it was found that the h(GLY2)GLP-2-induced increase in microvillus length required the IE-IGF-1R. Furthermore, IE-IGF-1R KO alone resulted in a significant decrease in microvillus length. Examination of the brush border membrane proteome as well as of whole jejunal mucosa demonstrated that villin was increased with h(GLY2)GLP-2 treatment in an IE-IGF-1R-dependent manner. Under both basal conditions and with h(GLY2)GLP-2 treatment of the IE-IGF-1R KO mice, changes in villin, IRTKS-1, harmonin, β-actin, and myosin-1a did not explain the decrease in microvillus length, in either the brush border or jejunal mucosa of KO animals. Collectively, these studies define a new role for the IE-IGF-1R within the microvillus, in both the signaling cascade induced by GLP-2, as well as endogenously.


Differential proteomic expression in isolated brush border membranes.
To understand the changes underlying the alterations in microvillus length, both with GLP-2 treatment and in the IE-IGF-1R KO mice, brush border membranes (BBM) were isolated from control and KO, vehicle-and h(GLY 2 )GLP-2-treated mice. Western blot demonstrated an 11.6-fold enrichment in the brush border protein, villin, as compared to the cytoplasmic protein, GAPDH (preliminary data, not shown). Mass spectrometry analysis identified over 1,200 proteins in the samples (Supplemental Table 1). Mouse Mines (www.mousemines.org) was used to identify the presence of ontologies within the category of "cellular component", including "cytoskeleton", "mitochondrial", "nuclear", "organelle", "membrane" and "microvillus". However, further analysis focused on the "actin filament" gene ontology (GO) category, which was significantly enriched (p < 0.05) and included a total of 71 known proteins, including those that are found in the microvillus. Protein expression levels were then determined for each group of animals relative to the vehicle-treated control mice (Fig. 3a). Marked differences in expression patterns were observed between vehicle-and h(GLY 2 )GLP-2-treated control and IE-IGF-1R KO mice, as well as between control and KO animals for each treatment group. To better understand these differential expression patterns, BBM structural proteins were categorized according to: 1) effects of GLP-2 that required the IE-IGF-1R; and 2) effects of the loss of the IE-IGF-1R alone. Select structural proteins, chosen for their roles in regulating microvillus length, were then analyzed in greater detail.   (e)). n = 5 for microvillus length; microvilli in 28 cells from 5 different villi were measured per mouse. n = 3 for packing angle; a minimum of 40 angles were taken per mouse. n = 3 for microvillus density in a 600 × 600 µm box. *P < 0.05 for the difference between treatment groups, # P < 0.05 for the difference from controls. Scale bar is 100 µm.
www.nature.com/scientificreports www.nature.com/scientificreports/ Villin. In the isolated BBM, VILLIN demonstrated a significant correlation (R 2 = 0.97; p < 0.05) for effects of h(GLY 2 )GLP-2 that require the IE-IGF-1R (category 1; Fig. 3b). VILLIN expression was increased to 1.43-fold in h(GLY 2 )GLP-2 treated control mice, with no change seen in h(GLY 2 )GLP-2 treated IE-IGF-1R KO animals. To further understand villin expression changes, the jejunal mucosa was analyzed in greater detail. Although Villin mRNA levels showed no difference between groups (Fig. 4a), changes in VILLIN protein levels, as determined by immunoblot, paralleled the BBM findings, with an 8.13-fold increase in the h(GLY 2 )GLP-2 treated control mice (p < 0.05; Fig. 4b). Immunofluorescence analysis of jejunal sections supported these findings (Fig. 4c), as control mice given h(GLY 2 )GLP-2 showed suggested increases in the intensity of VILLIN staining at the brush border. Furthermore, the GLP-2-induced increase in VILLIN required the IE-IGF-1R, as KO mice failed to show significant increases in protein expression as well as staining intensity.

IRTKS-1.
IRTKS-1 levels in the BBM demonstrated a significant correlation (R 2 = 0.93; p < 0.05) for effects of the IE-IGF-1R KO alone, being higher in both the vehicle (2.39-fold) and h(GLY 2 )GLP-2 treated (1.38-fold) KO mice, as compared to the control animals (category 2; Fig. 3b). Interestingly, although mRNA transcript levels were increased by GLP-2 treatment in both groups of animals (p < 0.05; Fig. 5a), the increase in IRTKS-1 in the IE-IGF-1R KO BBM was not observed by western blot of the jejunal mucosa. Instead, there was a 6.80-fold  www.nature.com/scientificreports www.nature.com/scientificreports/ increase in IRTKS-1 levels in the h(GLY 2 )GLP-2 treated control mice, and a smaller, non-significant increase in the KO animals (Fig. 5b). These changes were not observable under immunofluorescence, with the intensity and localization remaining unchanged (Fig. 5c).
Harmonin. In the BBM, HARMONIN also had a significant correlation (R 2 = 0.95; p < 0.05) for effects of the IE-IGF-1R KO alone, with 3.85-fold and 1.69-fold increases in expression in vehicle and h(GLY 2 )GLP-2 treated IE-IGF-1R KO mice, respectively (category 2; Fig. 3b). However, harmonin in the entire jejunal mucosa showed different results. Harmonin transcript levels were significantly decreased in the IE-IGF-1R KO vehicle mice, compared to those in the controls (p < 0.05; Fig. 6a), whereas the mucosa showed no change in HARMONIN expression in response to either h(GLY 2 )GLP-2 treatment or the KO (Fig. 6b). Furthermore, this trend was not apparent by harmonin immunostaining of the jejunum, as relative intensities and localization appeared similar between groups, with localization predominantly in the brush border (Fig. 6c). β-actin. In the BBM, β-ACTIN had a significant correlation (R 2 = 0.95; p < 0.05) for effects of the IE-IGF-1R KO alone (category 2; Fig. 3b). However, only the vehicle treated KO mice had higher β-ACTIN levels, with a 1.75-fold increase as compared to h(GLY 2 )GLP-2 treated KO mice, which demonstrated only a non-significant 1.18-fold increase. No changes in β-actin transcript levels were detected in the jejunal mucosa (Fig. 7a), while examination of protein levels in these tissue samples showed different results as compared to the isolated BBM, such that β-ACTIN showed a significant 4.3-fold increase in the jejunal mucosal of h(GLY 2 )GLP-2 treated control mice (p < 0.05; Fig. 7b). This finding was supported by immunostaining, where observable increases in fluorescent intensity were observed in h(GLY 2 )GLP-2 control mice, in both the brush border and basolaterally ( www.nature.com/scientificreports www.nature.com/scientificreports/ GLP-2 failed to demonstrate increases in protein expression, or in the intensity of the immunostaining in the brush border and basolaterally.

Myosin-1a.
Finally, in the BBM, MYO-1A did not show a significant correlation with either GLP-2 treatment or IE-IGF-1R KO (Fig. 3b). Analyses of the jejunal mucosa also failed to demonstrate any changes in jejunal mucosal expression of myo-1a at either the mRNA or protein levels (Fig. 8).

Discussion
Microvilli display uniform length, which is of functional importance 1 . Proteins within the BB region have therefore been studied to better understand their influence on microvillus length; however, the factors that regulate these proteins are poorly understood. The results of the present study indicate that the expression of several proteins with key roles in brush border structure is differentially regulated by the intestinotrophic hormone, GLP-2, as well as by one of its downstream signaling mediators, the IE-IGF-1R.
The BBM proteome has been isolated previously, and was reported to express approximately 650 identified proteins 2 . Unexpectedly, the isolation conducted herein contained over 1,200 proteins. Although the isolation protocols were identical, one key difference that may account for such a drastic difference in the number of identified proteins was the mass spectrometry instrument. The Orbitrap Fusion Lumos, used in the current study, has a faster scanning time and is more sensitive, as compared to the Thermo Scientific LTQ linear ion-trap utilized previously 2 , and therefore represents a technological advance given the almost ten-year time gap between the two proteomes. This may allow for the identification of previously unidentified brush border proteins, www.nature.com/scientificreports www.nature.com/scientificreports/ leading to enhanced understanding of the development and maintenance of this critical intestinal epithelial cell compartment.
The BBM isolation reported in this paper was validated in two ways. First, through western blot, probing for the ratio of VILLIN to GAPDH in both BBM and jejunal mucosa samples. VILLIN is a protein that, within the enterocytes, is only found in the brush border 34 ; thus, the ratio of VILLIN:GAPDH should be, and was found to be, higher in the BBM isolate. Another form of validation was conducted by analysis of the proteome using Mouse Mines, with the results showing protein categories as previously reported 2 , and significant up regulation of specific GO terms, under "cellular component" ontologies, that one would expect to find in the brush border, such as "brush border", "microvillus", "cluster of actin-based projections", and "actin filament bundles". h(GLY 2 )GLP-2 has been previously reported to increase microvillus length 23 , but how this is achieved is poorly understood. Through the use of the IE-IGF-1R KO mice, we have now shown that the increase in microvillus length with h(GLY 2 )GLP-2 treatment requires the IE-IGF-1R. One key microvillus protein that was regulated by the GLP-2 -IE-IGF-1R pathway was villin, an actin-bundling protein [35][36][37] , with a well-established role in epithelial wound repair 38 . Although it's deletion is not sufficient to abolish the formation of microvilli 5 , villin over-expression has been shown to induce the expression of membrane projections in cells that do not naturally do so 39 . In the present study, the protein but not the transcript levels of villin were increased with h(GLY 2 )GLP-2 treatment, in an IE-IGF-1R-dependent manner. Decreases in VILLIN levels have been reported previously in patients with Crohn's Disease, while mRNA levels also remained unchanged 40,41 . These findings suggest possible translational regulation of villin, as opposed to transcriptional modulation. Furthermore, with links to decreased IGF-1 levels in Crohn's disease patients 42  www.nature.com/scientificreports www.nature.com/scientificreports/ studies involving GLP-2 administration to intestinal epithelial villin-deficient animals, with and without induced enteritis, may prove useful to further interrogate this pathway.
Another interesting result was the lack of increase in the core-forming protein, β-actin, in the BBM of control mice treated with h(GLY 2 )GLP-2. With an increase in microvillus length, it would follow that there should be an increase in β-actin within the BBM, as this protein forms the core. However, the overall increase in microvillus length, although significant, was only 0.2 µm and, thus, may have been too small to detect an increase in β-actin expression. It has been shown that the ratio of β-actin to villin expression is 1:4 44 ; thus, increases in villin expression are more likely to be detected as compared to β-actin, within the BBM itself. However, when the entire jejunal mucosa was analyzed, there was a significant increase in β-actin expression with h(GLY 2 )GLP-2 treatment, which required the IE-IGF-1R. This result is also consistent with the immunostaining, which showed an increase in the intensity of the basolateral staining. Thus, the GLP-2 -IE-IGF-1R pathway is also important for regulating the expression of β-actin in the jejunal mucosa.
Unexpectedly, the IE-IGF-1R KO mice presented with shorter microvilli. The IE-IGF-1R has not been reported to play a role in regulating microvillus length, with IGF-1 and its receptor better known for their roles in whole body growth 45,46 , metabolism 47 , and cancer 48 . Focusing on the intestine, IGF-1 and its receptor are major determinants in colorectal cancer 49 and may be decreased in some patients with Crohn's disease 42 . IGF-1 signaling has also been shown to mediate the intestinotrophic effects of h(GLY 2 )GLP-2 17,29,31-33 , as well as to stimulate Na + / K + -ATPase activity and enhance Na + -coupled glucose absorption in enterocytes 50 . The present studies therefore define a new role for the IGF-1R in regulating the length of the intestinal brush border.
Decreases in microvillus length in response to BBM protein KO have been reported previously 8,9,11,13,14 , but these models also demonstrate changes in the actin core cytoplasmic projections (rootlets) 8 integrity of the plasma membrane 9 , and/or microvillus membrane 11 , hallmark features that were not present in the IE-IGF-1R KO mice. Plastin-1a KO mice have 20% shorter microvilli, but also lack rootlets, which was not observed with IE-IGF-1R KO 8 . Similarly, combinatory KO of villin, espin, and plastin-1a decreases both microvillus and rootlet length 14 . In contrast, KO of desmoplakin, a desmosomal protein, results in microvilli that are shorter but also misshaped, the latter of which was not seen in the IE-IGF-1R KO mice 51 . Harmonin KO animals also display shorter microvilli, in addition to changes in architecture and density that were not observed in the IE-IGF-1R KO 13 . Finally, KO of myo-1a results in longer, less densely packed microvilli, with extrusions of cellular cytoplasm in the intestinal lumen 9 , again, a phenotype that was not noted in the IE-IGF-1R KO model. Collectively, these findings indicate a unique role for the IGF-1R in regulating proteins that contribute to microvillus length without affecting microvillus density or structure. IRTKS-1 is known to play an important role in membrane bending and microvillus formation 3,52 and was recently reported to increase microvillus length during enterocyte differentiation 4 . IRTKS-1 may be regulated by the IE-IGF-1R, as the isolated BBM showed a significant correlation wherein the KO mice had higher expression levels than controls. What is still unclear is whether the IE-IGF-1R negatively regulates the expression of IRTKS-1, or if this protein is increased in the KO mice in compensation for the decrease in microvillus length. The jejunal mucosa analysis indicates the latter of the two possibilities. IRTKS-1 is not exclusively located in the BBM so, upon measuring the protein levels in the mucosa, they remained unchanged in the vehicle KO mice compared to vehicle controls, showing the IE-IGF-1R does not regulate overall expression of IRTKS-1. Furthermore, although h(GLY 2 )GLP-2 increased IRTKS-1 in the mucosa, with a smaller concentration of IRTKS-1 located in the BBM, the effects of GLP-2 were undetectable in this compartment. Instead, the BBM proteome showed a compensatory action of the enterocyte to elongate its microvillus. Interestingly, espin-8 expression in the BBM also showed increases in the IE-IGF-1R KO, as compared to the controls (data shown in Supplemental Table 1). Espin-8 has www.nature.com/scientificreports www.nature.com/scientificreports/ been reported to play a role in the effects of IRTKS-1 on the brush border 4 . The increase in espin-8 therefore supports a role for the increased IRTKS-1 in the KO mice as a compensatory attempt to enhance microvillus length.
The change in harmonin expression with the BBM may also indicate another compensatory change in the IE-IGF-1R KO to increase microvillus length. Harmonin levels were positively correlated with the effects of the KO alone, such that the KO mice, regardless of treatment, had higher levels of harmonin as compared to the control animals. Harmonin is known to be expressed primarily in the BBM 13 , as confirmed by the pattern of immunostaining, so it was surprising that the jejunal mucosa did not mirror this result. However, the differential sensitivities of the two methods must be considered and, therefore, their ability to detect changes in a protein which is expressed in relatively small amounts.
Myosin-1a is a protein that is exclusive to the brush border 53 and, accordingly, was found to be expressed in both the BBM and jejunal mucosa, with predominant localization to the brush border. However, myosin-1a is known to be important for membrane-actin cytoskeletal adhesion 9 and myosin-1a KO mice present with membrane extrusions and decreased microvillus density, hallmark features that were not seen here in response to either GLP-2 treatment or IGF-1R KO.
Although GLP-2 treatment is known to increase intestinal digestive and absorptive capacity 16,19,54,55 , KO of the GLP-2R reduces amino acid absorption but does not impact whole body growth 56 . It still remains unclear as to whether there are any functional consequences following IE-IGF-1R KO except for an inability to increase proliferation 29 or barrier function 17 in response to GLP-2 administration. However, no changes in body weight have been reported for the IE-IGF-1R KO mice 17,29 , as also found in the present study (data not shown). Notwithstanding, these studies have all been conducted for only two-weeks following induction of the KO which, given the relatively short time in additional to the small decrease in microvillus length, may not be sufficient to detect a robust change in body weight. Indeed, this is consistent with other KO models, wherein key brush border proteins are absent but no overall physiological effect is seen 5,8,9,13,14,57 . Consistent with this possibility, myo-1a, which was unaffected by IE-IGF-1R KO, has been shown to also be important in membrane trafficking within the microvillus 9 , and sucrase isomaltase and alkaline phosphatase were detected at normal levels in the BBM from the IE-IGF-1R KO (Supplemental Table 1), suggestive of normal trafficking.
Finally, one limitation of this study is the use of whole small intestine to prepare the brush border proteome, as compared to the molecular analyses of jejunal mucosa and full-thickness cross-sections. As the GLP-2 receptor is expressed at highest levels in the jejunum 24 , many studies have focused on this region of the gut 17,29,54,55 . However, as a consequence, changes in the proteome may also reflect differences in duodenal and/or ileal microvillar proteins that are not occurring in the jejunum. Further studies will be required to determine the possibility of site-specific differences in the regulation of microvillus length.
In conclusion, the results of the present study demonstrate not only that the IE-IGF-1R is essential for the GLP-2 induced increase in microvillus length, but that it also plays a key role in the maintenance of basal microvillus length under non-stimulated conditions. Although changes in several key microvillus proteins were demonstrated in response to experimental manipulation of these systems, the complex interplay of these proteins and the downstream mechanism(s) by which they regulate microvillus length remain to be fully elucidated.
Following induction, 0.1 μg/g h(GLY 2 )GLP-2 (American Peptide Company, Sunnyvale, CA) or vehicle (PBS) was injected sc q24hr for 11 d, with the final treatment given 3 hr prior to euthanasia 17,29,32,33 . The small intestine was collected and flushed with cold PBS. Multiple 2 cm mid-jejunal sections were then fixed in 10% formalin for 24 hr for paraffin embedding and sectioning (University Health Network, Toronto, ON), fixed in paraformaldehyde for 24 hr for frozen sectioning, fixed in 2.5% glutaraldehyde in Sorensen buffer for transmission electron microscopy (TEM), or frozen on dry ice and stored at −80 °C for mRNA and protein analysis; alternatively, the entire small intestine was collected for brush border membrane (BBM) isolation.
BBM isolation. As previously described 2  www.nature.com/scientificreports www.nature.com/scientificreports/ with 0.5 mM Pefabloc-SC (500 g, 8 min), and then washed twice in Solution A (75 mM KCl, 10 mM Imidazole, 1 mM EGTA, 5 mM MgCl 2 , and 0.02% Na-azide; pH 7.2). After resuspension of the pellet in Solution A, a 60% sucrose solution was added to make a final concentration of 50%. A 40% sucrose solution was layered on top, and the samples were spun at 146,900 g for 1.5 hr (Beckman Optima XPN 80, SW70Ti rotor). The interface between the two gradients, containing the isolated BBM was then removed and washed twice with solution A (500 g, 8 min), and stored at −80 °C. The isolated protein was quantified by BCA assay (Pierce BCA Assay, Thermo Scientific), and underwent western blot validation.
Mass spectrometry was performed by the SPARC BioCentre (The Hospital for Sick Children, Toronto, ON, Canada), using the Thermo Scientific Orbitrap Fusion-LumosTribid Mass Spectrometer (ThermoFisher, San Jose, CA) outfitted with a nanospray source and EASY-nLC 1200 nano-LC system (ThermoFisher, San Jose, CA) and equipped with ETD mode. Lyophilized peptide mixtures were dissolved in 0.1% formic acid and loaded onto a 75 μm × 50 cm PepMax RSLC EASY-Spray column filled with 2 μM C18 beads (ThermoFisher San, Jose CA) at a pressure of 900Bar and a temperature of 60 °C. Peptides were eluted over 240 min at a rate of 250 nL/min using a gradient set up as follows, where Buffer A is 0.1% formic acid and Buffer B is 80% acetonitrile, 0.1% formic Acid, all v/v in HPLC grade water: 0-100 min, 3-25%B; 100-228 min, 25-44%B; 228-230 min, 44-100%B; 230-240 min, 100%B.
Peptides were introduced by nano-electrospray into the Fusion-Lumos mass spectrometer (Thermo-Fisher). Data were acquired using the MultiNotch MS3 acquisition with synchronous precursor selection (SPS) with a cycle time of 5 sec. MS1 acquisition was performed with a scan range of 550 m/z-1800 m/z with resolution set to 120 000, maximum injection time of 50 msec and AGC target set to 4e5. Isolation for MS2 scans was performed in the quadrupole, with an isolation window of 0.7. MS2 scans were done in the linear ion trap with a maximum injection time of 50 msec and a normalized collision energy of 35%. For MS3 scans, HCD was used, with a collision energy of 30% and scans were measured in the orbitrap with a resolution of 50000, a scan range of 100 m/z-500 m/z, an AGC Target of 3e4, and a maximum injection time of 50 msec. The dynamic exclusion was applied using a maximum exclusion list of 500 with an exclusion duration of 20 sec.

Mouse mines analysis.
A total of 1,282 proteins were identified, and run through Mouse Mines (www. mousemines.org), returning a list of 1,223 genes. Parent ontologies, as well as significantly enriched gene ontologies, were determined. The significantly enriched "actin filament" gene ontology was used to narrow down the list to 71 proteins relevant to the brush border region. Protein spectral counts were then normalized to the highest count, giving new values ranging from 0.01-1.0. These normalized values were then analyzed by Pearson correlations against models which showed differential expression between groups. A change was defined as any normalized value which was ≥0.3 compared to other groups, and no change between groups was defined as having a ≤ 0.1 difference in normalized expression, with 0.5 as the baseline.
Microscopy. Crypt-villus height was measured on hematoxylin/eosin-stained slides, with a minimum of 20 villi and crypts per mouse to make n = 1 (AxioVision software, Carl Zeiss, Canada). For immunofluorescent staining, one mouse from each treatment and each genotype group was represented on each slide. After xylene dewaxing and rehydration for paraffin-embedded slides, or warming up to room temperature for frozen slides, they underwent heat-induced antigen retrieval, using citrate buffer for VILLIN, and Tris-EGTA for MYOSIN-1A, β-ACTIN, HARMONIN and IRTKS-1 (Baiap2l1). Tissues were blocked in 10% normal goat serum, and incubated with primary antibodies (Supplemental Table 2) overnight at 4 °C. Secondary antibodies (Supplemental Table 2) were added for 1 hr, in the dark, at room temperature. An AxioPlan epifluorescence microscope (Carl Zeiss, Canada) was used for all imaging, with constant exposure times used for all 4 samples per slide. Z-stacking and deconvolution was conducted using the AxioVision software when required. Negative controls were run by omission of all primary antisera (Supplemental Fig. 6).
For TEM, glutaraldehyde fixed tissues were post-fixed in 2% phosphate-buffered OsO 4 , followed by dehydration in acetone, and embedding and polymerization in Epon-Araldite (Hospital for Sick Children, Toronto, Ontario, Canada). For determining microvillus and terminal web length, a minimum of 4 measurements was taken for each cell for a total of 28 cells from at least 5 different villi per mouse to make n = 1, on a Hitachi H-7000 electron microscope. Microvillus length was determined, using the Hitachi software, by measuring from the distal tip to the plasma membrane, and the terminal web was measured from the plasma membrane to the end of cytoplasmic projection. Packing angle was determined by taking a minimum of 40 measurements of the angle between three adjacent microvillus cross sections, to make n = 1 for each mouse. Microvillus density was determined as the number of intact microvillus cross-sections within a 3.6 × 10 8 µm 2 box, for n = 3 mice per group. RNA isolation and real-time quantitative reverse-transcription polymerase chain reaction (q-RT-PCR). Total RNA was isolated from jejunal mucosal scrapes using the RNeasy kit with QiaShredder (Qiagen, Inc, Mississauga, ON, Canada), converted into complementary DNA (5x all-in-one RT Master Mix, abm), and assessed by Taqman Gene Expression Assay (Applied Biosystems, Foster City, CA), using TaqMan primers as shown in Supplemental Table 3). The delta-delta CT method was used to normalize relative mRNA levels 60 . Western blot. Jejunal mucosal scrapes were sonicated in RIPA lysis buffer, and protein contained in BBM isolates and mucosal scrapes was quantified by BCA assay (Pierce BCA Assay, Thermo Scientific). Proteins were run on an 8% PAGE gel, transferred onto a PVDF membrane, and incubated with primary antibodies overnight at 4 °C (Supplemental Table 4). Secondary antibodies were incubated for 1 hr, at room temperature (Supplemental Table 4), and visualized using ECL detection reagent (Cell Signaling Technology). Samples were normalized to small intestinal controls where appropriate, and all samples were normalized to vehicle-treated control mice. Each